Endostatin fragments and variants for use in treating fibrosis

ABSTRACT

Materials and methods for using polypeptides containing fragments and variants of endostatin to treat fibrosis are described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from U.S. ProvisionalApplication No. 62/171,889, filed on Jun. 5, 2015, and U.S. ProvisionalApplication No. 62/257,607, filed Nov. 19, 2015.

TECHNICAL FIELD

This document relates to materials and methods for treating fibrosis,and particularly to the use of polypeptides containing fragments andvariants of endostatin for treating fibrosis.

BACKGROUND

Endostatin, a 183 amino acid proteolytic cleavage fragment correspondingto the C-terminus of collagen 18, has anti-tumor activity with no toxicside effects (O'Reilly et al. (1997) Cell, 88: 277-285; Kisker et al.(2001) Cancer Res, 61:7669-7674; Dhanabal et al. (1999) Cancer Res, 59:189-197; Yoon et al. (1999) Cancer Res, 59: 6251-6256; Folkman andKalluri, (2003) Cancer Medicine, 6th edition, pp. 161-194. Hamilton: B.C. Decker Inc.). A number of anti-angiogenic activities have beenreported for this protein, such as inhibition of endothelial cellproliferation, migration, and tube formation. This activity has beenlocalized to the N-terminal region of endostatin. Endostatin alsosuppresses vascular endothelial growth factor (VEGF)-induced vascularpermeability (Takahashi et al. (2003) Faseb J, 17: 896-898). Endostatininhibits endothelial cell migration by inhibiting phosphorylation offocal adhesion kinase via binding to α5β1 integrin (Wickstrom et al.(2002) Cancer Res, 62: 5580-5589). It also has been shown that cellsurface glypicans are low-affinity endostatin receptors (Karumanchi etal. (2001) Mol Cell, 7: 811-822). Endostatin has been implicated inseveral signaling pathways, such as downregulation of c-myc (Shichiriand Hirata (2001) Faseb J, 15: 1044-1053), cyclin-D1 (Hanai et al.(2002) J Biol Chem, 277. 16464-16469) and RhoA activity (Wickstrom etal. (2003) J Biol Chem, 278: 37895-37901), blockage of VEGF signaling(Hajitou et al. (2002) Faseb J, 16: 1802-1804; Kim et al. (2002) J BiolChem, 277: 27872-27879), and inhibition of the wnt-signaling pathway(Hanai et al. (2002) J Cell Biol, 158: 529-539). Further, endostatin hasbeen shown to bind and inactivate metalloproteinases (Kim et al. (2000)Cancer Res, 60: 5410-5413; Nyberg et al. (2003) J Biol Chem, 278:22404-22411; Lee et al. (2002) FEBS Lett, 519: 147-152) and to regulatea spectrum of genes which suppress angiogenesis (Abdollahi et al. (2004)Mol Cell, 13: 649-663).

The crystal structures of both murine and human endostatin have beenresolved (Hohenester et al. (1998) Embo J, 17: 1656-1664; Ding et al.(1998) Proc Natl Acad Sci USA, 95: 10443-10448), and show anoncovalently held dimer at high concentration required forcrystallization (Ding et al. (1998) Proc Natl Acad Sci USA, 95:10443-10448). The presence of two disulfide bonds results in a highlyfolded structure. Endostatin binds one atom of zinc per monomer viathree histidines in the N-terminus of the molecule (histidines 1, 3, and11) and aspartic acid 76. The heparin binding property of endostatin ismediated by noncontiguous arginines clustered over the three dimensionalglobular surface of the molecule (Sasaki et al. (1999) Embo J, 18:6240-6248).

Excessive deposition of extra cellular matrix (ECM) components such asfibronectin (FN) and type I collagen (Col1α1) by organ fibroblasts isdefined as fibrosis. Organ fibrosis is the final common pathway for manydiseases that result in end-stage organ failure. However, effectivetherapy for organ fibrosis is still unavailable (see, for example,Bjoraker et al., Am. J. Respir. Crit. Care. Med 2000; 157:199-203).Uncontrollable wound-healing responses, including acute and chronicinflammation, angiogenesis, activation of resident cells, and ECMremodeling, are thought to be involved in the pathogenesis of fibrosis(Wynn, J Clin Invest 2007; 117:524-29; Kalluri et al., Curr Opin NephrolHypertension 2000; 9:413-8). TGF-β is the prototype fibrotic cytokinethat is increased in fibrotic organs, and contributes to the developmentof fibrosis by stimulating the synthesis of ECM molecules, activatingfibroblasts to α-smooth muscle actin (α-SMA)-expressing myofibroblasts,and downregulating matrix metalloproteinases (MMPs) (see, for example,Branton et al., Microbes Infect 1999; 1:1349-65). Despite highexpectations, a clinical trial of a monoclonal anti-TGF-β antibody inpatients with early systemic sclerosis (SSc) failed to show any efficacy(Varga et al., Nature Reviews Rheumatology 2009; 5:200-6). Thus, a needremains for other treatments of fibrosis.

SUMMARY

This document is based, at least in part, on the development ofC-terminal endostatin fragments and variants thereof that haveanti-fibrotic activity, as well as the development of methods forproducing such fragments and variants in plants, and methods for usingthe fragments and variants to treat fibrosis in subjects in needthereof.

This document also is based, at least in part, on the discovery that anendostatin-based polypeptide containing the E3 region of endostatin(amino acids 133-180 of SEQ ID NO:2 and SEQ ID NO:13, for example)linked to an Ig-Fc polypeptide can form high molecular weight (HMW)multimers, unlike typical peptide-Fc fusion proteins. The HMW form canprovide advantages in purification (e.g., by ultrafiltration ortangential flow filtration (TFF)), and may also be used in the treatmentof fibrosis.

In one aspect, this document features an isolated polypeptide, whereinthe polypeptide comprises SEQ ID NO:14 and has anti-fibrotic activitywhen administered to a subject in need thereof. The isolated polypeptidecan further include a secretory sequence, a peptide tag (e.g., a 6Histag, a tag containing a KDEL polypeptide, or both), Ala-Ser-Lys sequenceat the C-terminal end of SEQ ID NO:14, and/or an IgG Fc domain (e.g., anIgG1 Fc domain, such as a human IgG1 Fc domain), amino acids 27 to 43 ofSEQ ID NO:17. The polypeptide can comprise SEQ ID NO:16 or SEQ ID NO:17.The polypeptide can be a high molecular weight multimer.

In another aspect, this document features an isolated polypeptide,wherein the polypeptide (a) comprises amino acids 133 to 180 of SEQ IDNO:2 and one or more of a secretory sequence, a peptide tag, a KDELsequence, an Ala-Ser-Lys sequence at the C-terminal end of amino acids133 to 180 of SEQ ID NO:2, an IgG Fc domain, and/or amino acids 27 to 43of SEQ ID NO:17; and (b) has anti-fibrotic activity when administered toa subject in need thereof. The polypeptide can comprise SEQ ID NO:15.

In another aspect, this document features an isolated polypeptide,wherein the polypeptide (a) comprises amino acids 133 to 180 of SEQ IDNO:13 and one or more of a secretory sequence, a peptide tag, a KDELsequence, an Ala-Ser-Lys sequence at the C-terminal end of amino acids133 to 180 of SEQ ID NO:2, an IgG Fc domain, and/or amino acids 27 to 43of SEQ ID NO:17; and (b) has anti-fibrotic activity when administered toa subject in need thereof.

In another aspect, this document features an isolated polynucleotideencoding a polypeptide as disclosed herein, where the polynucleotide isoperably linked to a heterologous promoter. This document also featuresan expression vector containing the isolated polynucleotide. Theexpression vector can be a launch vector (e.g., a viral launch vector).In addition, this document features an Agrobacterium tumefaciens cellcomprising the expression vector.

In another aspect, this document features a high molecular weightmultimer comprising SEQ ID NO:14 and having anti-fibrotic activity whenadministered to a subject in need thereof.

In still another aspect, this document features a pharmaceuticalcomposition containing (a) a polypeptide and/or multimers, as describedherein, and (b) a pharmaceutically acceptable carrier.

This document also features a method for making a polypeptide havinganti-fibrotic activity as disclosed herein. The method can include (a)introducing into a plant a plant viral vector that includes apolynucleotide encoding the polypeptide of claim 1, claim 12, claim 13,or claim 15 having antifibrotic activity; and (b) maintaining the plantunder conditions and for a time sufficient that the polynucleotide isexpressed in at least some plant cells. In some embodiments, the methodcan include (a) introducing into a plant (i) a carrier vector thatincludes a functional coat protein encoding component from a first plantvirus, and (ii) a producer vector that includes a polynucleotideencoding the polypeptide having antifibrotic activity and at least onecomponent from a second plant virus, but lacks a functional coat proteingene; or (i) a carrier vector that includes a functional movementprotein encoding component from a first plant virus, and (ii) a producervector that includes a polynucleotide encoding the polypeptide havingantifibrotic activity and at least one component from a second plantvirus but lacks a functional movement protein gene; or (i) a carriervector that includes a functional coat protein encoding component from afirst plant virus, and (ii) a producer vector that includes apolynucleotide encoding the polypeptide having antifibrotic activity andat least one component from a second plant virus but lacks one or morefunctional replication protein genes normally found in the second plantvirus; (b) maintaining the plant under conditions and for a timesufficient to allow the carrier vector to complement the producervector, so that the producer vector moves systemically in the plant; and(c) maintaining the plant under conditions and for a time sufficientthat the polynucleotide is expressed in at least some plant cells.

The introducing step can include vacuum infiltration. The method canfurther include harvesting the plant, wherein the plant comprises thepolypeptide having anti-fibrotic activity; extracting the polypeptidehaving anti-fibrotic activity from the plant; and/or purifying thepolypeptide having anti-fibrotic activity.

In another aspect, this document features a method of treating a subjectwith fibrosis. The method can include selecting a subject with fibrosis,and administering to the subject a therapeutically effective amount of apolypeptide as described herein, thereby treating the subject withfibrosis. The subject can have a pulmonary fibrosis. The method canfurther include administering to the subject a therapeutically effectiveamount of another therapeutic agent (e.g., a therapeutic agent selectedfrom the group consisting of corticosteroids, immunosuppressive agents,acetylcysteine, d-penicillamine, colchicine, Relaxin, steroids,cyclosporine, methotrexate, cyclophosphamide, azathioprine,mycophenolate, glitazones, endothelin receptor antagonists, andFulvestrant). The corticosteroid can be prednisone. Theimmunosuppressive agent can be methotrexate or cyclosporine. Theadministering can include oral administration or intravenousadministration.

In yet another aspect, this document features a composition comprisingcontaining a pharmaceutically acceptable carrier and a polypeptide asdescribed herein, wherein the composition is formulated for oral orintravenous administration to a subject. The polypeptide can include SEQID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1F. ECM production in recombinant endostatin (rE)- andendostatin-derived peptide-treated fibroblasts in combination with TGF-βstimulation. FIG. 1A: FN and Col1α1 expression in human normal lungfibroblasts (NL) treated with vehicle (V), rE alone, or both with priorTGF-β stimulation. Proteins were detected with a Western blot.Glyceraldehyde e-phosphate dehydrogenase (GAPDH) was used as a loadingcontrol for lysates. FIG. 1B: FN and Col1α1 expression of endostatinpolypeptide-treated lung fibroblasts following TGF stimulation inprimary pulmonary fibroblasts from a healthy control, a patient withSSc, and a patient with IPF. FIG. 1C: Graphical summary of FN and Col1α1expression in lung fibroblasts obtained using fibroblasts from 4 healthycontrols (NL), 3 patients with SSc, and 3 patients with IPF. Intensityof bands was normalized to that of GAPDH and expressed as a ratio toVehicle (V). Paired-t test was used for statistical analysis. *P<0.04,**P<0.01. FIG. 1D: Representative result of FN and Col1α1 levels inhuman skin fibroblasts obtained from a patient with morphea and apatient with SSc. FIG. 1E: Representative result of FN and Col1α1expression in fibrotic fibroblasts obtained from a patients with IPFtreated with V, 5 μg/ml of rE, or endostatin polypeptides alone (left).IPF fibroblasts were treated with different concentrations (5, 10, and20 μg/ml) of E4. Dimethyl sulfoxide (DMSO) (V) was added in a volumeequivalent to that in the lane corresponding to 20 μg/ml of E4 (right).FIG. 1F: α-SMA levels in normal lung fibroblasts treated with endostatinpolypeptides following TGF-β stimulation.

FIGS. 2A-2C. Ex vivo human skin fibrosis organ culture model. FIG. 2A:Recombinant TGF-β or 1×phosphate buffered saline (PBS) (vehicle) wasinjected intradermally into human skin explants at a concentration of 1,10, 50 ng/ml. Skin was harvested 1 week post-injection. RepresentativeH&E (hematoxylin and eosin stain) images are shown in the upper row, andimages of Masson trichrome-stained section are shown in the lower row.Magnification, 20×. FIG. 2B: Recombinant endostatin (rE) was injectedinto human skin at a concentration of 1, 5, 10 μg/ml. 1×PBS was used asa vehicle control (V). Representative H&E images are shown.Magnification, 20×. FIG. 2C: Endostatin polypeptides (E-1, E-2, E-3, andE-4; all at 10 μg/ml) were injected intradermally in human skin. DMSOwas used as a vehicle control (V). Representative H&E images are shown.Magnification, 20×.

FIGS. 3A-3B. The effect of recombinant endostatin on TGF-β-inducedfibrosis and dermal thickness in human skin. FIG. 3A: Representative H&Eimages of human skin injected with Vehicle, 10 ng/ml TGF-β alone, or rE(1, 5, and 10 μg/ml) in combination with TGF-β (10 ng/ml). Tissues wereharvested one week post-injection. Magnification, 20×. FIG. 3B:Graphical presentation of dermal thickness. Data represent fourindependent experiments in triplicate using human skin explants fromfour different donors. Mann-Whitney U test was used for statisticalanalysis. *P<0.04.

FIGS. 4A-4B. The effect of endostatin polypeptides TGF-β-inducedfibrosis and dermal thickness in human skin. FIG. 4A: Representative H&Eimages of human skin injected with Vehicle, 10 ng/ml TGF-β alone, orE-1, E-2, E-3, or E-4 (10 μg/ml) in combination with TGF-β (10 ng/ml).Magnification, 20×. FIG. 4B: Graphical presentation of dermal thicknessdata shown in FIG. 4A. Data represent two independent experiments usinghuman skin explants from two donors, and each experiment was done intriplicate. Mann-Whitney U test was used for statistical analysis.*P<0.05, **P<0.02.

FIGS. 5A-5B. Dose response of E-1 and E-4 in TGF-β-induced fibrosis.FIG. 5A: Representative H&E images of human skin injected with E-1(upper row) or E-4 (lower row) at a concentration of 1, 5, 10, and 20μg/ml in the presence of TGF-β (10 ng/ml). Magnification, 20×. FIG. 5B:Graphical analysis of dermal thickness data shown in FIG. 5A. DMSO wasused as a vehicle control. Experiments were conducted in duplicate, anddermal thickness was measured in 6 fields from each section.Mann-Whitney U test was used for statistical analysis. *P<0.02,**P<0.01.

FIGS. 6A-6B. The effect of endostatin polypeptides in the development offibrosis in vivo in mouse skin. FIG. 6A: Mice were injectedintradermally with vehicle, 10 ng/ml TGF-β alone, or E-1, E-2, E-3, andE-4 (10 μg/ml) in combination with TGF-β (10 ng/ml). Skin was harvestedafter 1 week post-injection. Sections were stained with H&E.Magnification, 20×. FIG. 6B: Graphical summary of dermal thickness datashown in FIG. 6A. Data represent four independent experiments, each donein duplicate. Mann-Whitney U test was used for statistical analysis.*P<0.04, **P<0.01.

FIGS. 7A-7B. Capacity of endostatin polypeptide to inhibit tubularformation in MATRIGEL®. FIG. 7A: Representative images of MATRIGEL®cultures of HUVECs treated with vehicle, rE (50 nM), or E4 (50 nM). Anequivalent amount of DMSO was used as vehicle. Magnification 40×. FIG.7B: Image quantification of the cord formation shown in FIG. 7A. Datashown summarize results of three independent experiments. *P<0.05,one-way ANOVA followed by Bonferroni's test.

FIGS. 8A-8B. The effect of endostatin E-4 on bleomycin induced dermalfibrosis in vivo. FIG. 8A: Mice were injected subcutaneously with 1×PBSas vehicle (V) or Bleomycin (Bleo; 20 μg/mouse) daily. E-4 (10 μg/ml)was mixed with bleomycin on the first day, and daily bleomycinadministration was continued without subsequent injections of E4(Bleo+E-4). Skin was harvested after 10 days. Sections were stained withH&E. Magnification, 100×. FIG. 8B: Graphical summary of dermal thicknessdata shown in FIG. 8A. Data represent three independent experiments.Mann-Whitney U test was used for statistical analysis. *P<0.001,**P<0.00001. E4 administration caused a significant attenuation ofbleomycin induced dermal fibrosis even with a single administration ofE4.

FIG. 9. E4 reverses TGF β-induced dermal fibrosis even if administered 3days following TGF-β. Mouse skin was treated with TGF-β day 1 and E-4Lor E-1L (this is E-4 and E-1 administered after a 3 day lag betweenadministration of the fibrotic trigger and the administration of thepeptide. E-1 or E-4 was administered intraperitoneally (IP) at day 3 andharvested at day 7. E4 caused a significant decrease of TGF-β induceddermal fibrosis on day 7. Thus E4 prevents (FIGS. 4-6) and reverses(FIG. 9) dermal fibrosis triggered by TGF-β.

FIG. 10A-10C. E4 attenuates bleomycin induced lung fibrosis in vivo.FIG. 10A: Sixty μg of bleomycin was administrated intratracheally incombination with DMSO as a vehicle (Bleo) or E-4 (Bleo+E-4; 10 μg/ml).In some mice, E-4 (10 μg/ml) was administered intratracheally (IT) threedays following bleomycin treatment (Bleo+E-4L). PBS was used as avehicle for bleomycin (V). Lungs were harvested 10 days post-treatment.Representative images stained with H&E (left panel) and Masson trichrome(right panel) are representative of 3 independent experiments.Magnification, 100×. E4 administered concomitantly with bleomycin orthree days following bleomycin caused a marked reduction in fibrosis andMasson Trichrome staining. FIG. 10B: Quantification of acid solublecollagen obtained from mouse lungs treated with V. Bleo, Bleo+E-4, andBleo+E-4L. The levels of collagen are presented as μg/mg (lung) fromthree independent experiments. Unpaired-t test was used for statisticalanalysis. *P<0.05. E4 polypeptide given 3 days after bleomycinsignificantly reduced collagen levels in mouse lungs. FIG. 10C: Lowermagnification (2×) of mouse lung shown in FIG. 9 (BLM+E4/E4L IT day 10).For Bleo+E4L, Bleo was administered first, then there was a lag of threedays between Bleo and E4 administration).

FIG. 11. E4 attenuates bleomycin induced lung fibrosis in vivo whetheradministered intraperitoneally (IP) or intratracheally (IT). Bleomycinwas administered IT at day 1, and E4 was administered either IP or IT atday 3. Lungs were harvested at day 21. E4 caused a significantattenuation of bleomycin induced lung fibrosis on day 21 whetheradministered IP or IT. Thus E4 is effective at reducing fibrosisirrespective of the route of administration. Results are shown forvehicle alone (V), bleomycin alone (Bleo), bleomycin and E4 administeredIP, and bleomycin and E4 administered IT.

FIG. 12. E4 reduces fibrosis in vivo by reducing levels of lysyl oxidase(LOX), thus reducing crosslinking of collagen and rendering it lessstable and more susceptible to proteolytic degradation. Lung sections ofmice treated with BLM with or without E4 were used inimmunohistochemistry to detect LOX. The sections shown are control IgG,phosphate buffered saline, bleomycin and bleomycin followed by treatmentwith E4.

FIG. 13. E4 reduces fibrosis in vitro by blocking TGF-β-induced LOXproduction in primary human lung fibroblasts. Normal lung fibroblasts inpassage 4 were treated with vehicle, E4, TGF-β, or TGF-β followed 30min. later by E4. Media conditioned by the fibroblasts were analyzedusing Western blot analysis after 48 hour. Lane 1: Vehicle (DMSO); Lane2: E-4; Lane 3: TGF-β; Lane 4: TGF-β followed by E4. Similar resultswere obtained when LOX mRNA levels were examined by real-time PCR.

FIG. 14A-14B. FIG. 14A: E4 reduces fibrosis in vitro by inducing MMP-2activity in primary human lung fibroblasts, thus resulting in increaseddegradation of collagen and other matrix proteins. Digital image of agelatin zymography gel showing increased MMP-2 activity when primaryhuman lung fibroblasts are treated with E-4 following TGF-β (lane 4).Lane 1: Vehicle (DMSO); Lane 2: E-4; Lane 3: TGF-β; Lane 4: TGF-βfollowed by E4. FIG. 14B: Digital image showing that both total andactive MMP-2 levels are increased in cells treated with TGF-β and E-4.This suggests E-4 increases levels of MMP-2 pro-enzyme, but alsoincreases levels of active matrix metalloproteinase (MMP-2, also calledmetalloprotease-2).

FIG. 15. E4 reduces fibrosis in vitro by inducing expression of ID1, aninhibitor of TGF-β, in primary human lung fibroblasts. Real-time PCRanalysis was performed to determine the ID1 mRNA levels under theindicated conditions.

FIG. 16. E-4 reduces fibrosis in vitro by reducing levels of the mastertranscription factor Egr-1 in primary human lung fibroblasts. Reductionof Egr-1 levels parallels that of collagen, SMA, and fibronectin. Lane1: vehicle (DMSO); Lane 2: E4; Lane 3: TGFβ, Lane 4 TGFβ followed by E4after 60 minutes. The samples were harvested after 24 hours.

FIG. 17A-17B. The effect of endostatin peptides on established fibrosistriggered by TGF-β in human skin. FIG. 17A: Vehicle (DMSO), E-1, or E-4(10 mg/ml) was additionally injected to human skin 2 dayspost-administration of 10 ng/ml TGF-β (V, E-1L and E-4L, respectively).Representative H&E images of human skin were shown. Magnification, 20×.FIG. 17B: Graphical presentation of dermal thickness data shown in A.Data represent two independent experiments using human skin explantsfrom two donors, and each experiment was done in duplicate. Mann-WhitneyU test was used for statistical analysis. *P<0.01.

FIG. 18. Purification scheme for untagged E3 based on differentialsolubility.

FIG. 19. E3 variants produced in plants.

FIG. 20. Oral Plant-Made Pharmaceutical (PMP) E3 variants preventbleomycin-induced pulmonary fibrosis.

FIG. 21. Intravenous dosing of E3-Fc is comparable to oraladministration.

FIG. 22. Intravenous E3-Fc has the potential to reverse lung fibrosis.

FIG. 23. Expression of human IgG Fc fusions to E3 in a schematic (leftpanel) and a Western blot (right panel).

FIG. 24. Purification of human IgG Fc fusions to E3.

FIG. 25. Human IgA1, IgA2, and IgM Fc fusions to E3 are insoluble orpoorly expressed.

FIG. 26. Amino acid sequences of E3-Fc (SEQ ID NO:16), E3 (SEQ IDNO:20), and E4 (SEQ ID NO:21).

FIG. 27. iBio-CFB03 drug substance manufacturing process block diagram.

FIG. 28. Schematic of the pGR-D4 vector for expression of iBio-CFB03.

FIG. 29. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) of iBio-CFB03 Purified by Protein A and TFF.

FIG. 30. Size exclusion chromatogram of TFF Purified iBio-CFB03.

FIG. 31. Cation exchange (CEX) chromatogram of iBio-CFB03.

FIG. 32. SDS-PAGE gel image of iBio-CFB03 CEX fractions.

FIG. 33. Size exclusion chromatograms of CEX fractions 1A4 (top) and 1B2(bottom).

FIG. 34. Size Exclusion High Performance Liquid Chromatography (SE-HPLC)chromatograms of non-reduced iBio-CFB03 at 220 nm.

FIG. 35. Matrix Assisted Laser Desorption/Ionization Time-of-Flight MassSpectrometry (MALDI-TOF MS) spectrum of reduced iBio-CFB03.

FIG. 36. SDS-PAGE gel image of iBio-CFB03 under reduced (R) andnon-reduced (NR) conditions.

FIG. 37. iBio-CFB03 sequence with tryptic peptide fragments highlightedfor correlation with FIG. 38.

FIG. 38. MALDI-TOF MS spectrum of in-gel tryptic digested iBio-CFB03band 1 (reduced).

FIG. 39. Schematic for oral and IV administration in a bleomycin model.

FIG. 40. Hematoxylin and eosin stain of mouse lung treated with oral andIV administration of iBio-CFB03.

FIG. 41. Hydroxyproline assay of mouse lung in bleomycin model treatedwith oral and IV iBio-CFB03.

FIG. 42. Hydroxyproline assay of mouse lung in repeat experiment ofbleomycin model treated with oral and IV iBio-CFB03.

FIG. 43. Hematoxylin and eosin stain of mouse skin treated with osmoticpump subcutaneous (s.c.) administration of iBio-CFB03.

FIG. 44. Skin thickness of mice treated with osmotic pump s.c.administration of iBio-CFB03.

FIG. 45. Hydroxyproline assay of human skin in TGF-β model treated withiBio-CFB03.

FIG. 46. Schematic of mechanism of action for E4 endostatin peptide.

FIG. 47. SDS-PAGE gel of E3 Fc fusions E3-Fc (END-25), E3-linker-Fc(END-26), and C67A E3-Fc (END-55) lysates imaged for total protein (leftpanel), and Western blotted with an anti-Fc antibody by Western blot(right panel).

FIG. 48. END-55 Simulated Gastric Fluid (pepsin) digestion. SDS-PAGE gelof total protein (left panel), and Western blotted with either a chickenanti-E3 antibody (middle panel) or anti-human Fc antibody (right panel).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile [8123-84102-03 Sequence Listing.txt, Apr. 4, 2012, 12.5 kilobytes],which is incorporated by reference herein. In the accompanying sequencelisting:

SEQ ID NO:1 is an exemplary nucleic acid sequence encoding humanendostatin.

SEQ ID NO:2 is the amino acid sequence of human endostatin.

SEQ ID NO:3 is an exemplary nucleic acid sequence encoding mouseendostatin.

SEQ ID NO:4 is the amino acid sequence of mouse endostatin.

SEQ ID NO:5 is an exemplary nucleic acid sequence encoding a humanimmunoglobulin (Ig)G1 protein.

SEQ ID NO:6 is the amino acid sequence of a human IgG1 protein.

SEQ ID NO:7 is an exemplary nucleic acid sequence encoding a linker.

SEQ ID NO:8 is an amino acid sequence of a linker.

SEQ ID NO:9 is a portion of the rat endostatin polypeptide.

SEQ ID NO:10 is a portion of the cow endostatin polypeptide.

SEQ ID NO:11 is a portion of the human collagen XV polypeptide.

SEQ ID NO:12 is an exemplary nucleic acid sequence encoding anendostatin.

SEQ ID NO:13 is an amino acid sequence of an exemplary endostatinpolypeptide that differs from SEQ ID NO:2 by three amino acidsubstitutions.

SEQ ID NO:14 (SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAY IVLAIENSFMT) is aportion of human endostatin having a C→A mutation at the underlinedposition.

SEQ ID NO:15 is an exemplary amino acid sequence including a secretorysequence, a portion of human endostatin, and a peptide tag.

SEQ ID NO:16 is an exemplary amino acid sequence including a secretorysequence, a portion of human endostatin having a C67A mutation, and anIgG Fc domain.

SEQ ID NO:17 is an exemplary amino acid sequence including a secretorysequence, a portion of collagen XVIII, a portion of human endostatinhaving a C67A mutation, and a peptide tag.

SEQ ID NO:18 (QKSVWHGSDPNGRRLTE) is a 17 amino acid portion of collagenXVIII.

SEQ ID NO:19 is a four-amino acid endoplasmic reticulum targetingsequence.

SEQ ID NO:20 is the amino acid sequence of the E3 portion of humanendostatin.

SEQ ID NO:21 is the amino acid sequence of the E4 portion of humanendostatin.

SEQ ID NO:22 is the amino acid sequence of an E3_C67A-Fc_IgG2 fusion.

SEQ ID NO:23 is the amino acid sequence of an E3_C67A-Fc_IgG3 fusion.

SEQ ID NO:24 is the amino acid sequence of an E3_C67A-Fc_IgG4 fusion.

SEQ ID NO:25 is the amino acid sequence of an E3_C67A-Fc_IgA1 fusion.

SEQ ID NO:26 is the amino acid sequence of an E3_C67A-Fc_IgA2 fusion.

SEQ ID NO:27 is the amino acid sequence of an E3_C67A-Fc_IgM fusion.

SEQ ID NO:28 is the amino acid sequence of the J-Chain_PVX_sgp36.

SEQ ID NO:29 is the amino acid sequence of an IgG1_Fc-E3_C67A C-terminalfusion.

DETAILED DESCRIPTION

C-terminal endostatin polypeptides are provided herein. In someembodiments, these polypeptides include, consist essentially of, orconsist of (1) at least 40 consecutive amino acids of the amino acidsequence set forth as amino acids 133-180 of SEQ ID NO:13 or SEQ IDNO:2; (2) at least 40 consecutive amino acids of the amino acid sequenceset forth as amino acids 133-180 of SEQ ID NO:13 or SEQ ID NO:2, with atmost 5 amino acid substitutions, (3) the amino acid sequence set forthas amino acids 133-180 of SEQ ID NO:13 or SEQ ID NO:2; (4) the aminoacid sequence set forth as amino acids 133-180 of SEQ ID NO:13 or SEQ IDNO:2 with at most 5 amino acid substitutions; or (5) the amino acidsequence set forth as SEQ ID NO:14; wherein the polypeptide hasanti-fibrotic activity and wherein the polypeptide does not includeamino acids 1-92 of SEQ ID NO:13 or SEQ ID NO:2. In some embodiments,the polypeptide is amidated at the C-terminus. Methods of making thesepolypeptides also are provided. In some embodiments, the polypeptidescan be produced in plants. In addition, polynucleotides encoding thepolypeptides, host cells transformed with the polynucleotides, andmethods of using the polypeptides and polynucleotides are provided. Themethods can include the treatment of fibrosis in a subject. For example,methods are provided for treating fibrotic conditions of the lung andthe skin, as well as other organs (e.g., liver cirrhosis, cornealfibrosis, and kidney fibrosis). In some embodiments, the anti-fibroticC-terminal endostatin polypeptides disclosed herein can selectivelyinhibit fibrosis. In some examples, fibrosis is inhibited withoutinhibiting angiogenesis. Thus, the C-terminal endostatin polypeptidescan be used to more specifically and selectively target unwantedfibrosis, without interfering with angiogenesis, to achieve a desiredtherapeutic outcome.

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: A Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Amidation or amide derivative: A post-translational modification to forman amide that can enhance the biological activity of the polypeptide. Inamidation, the C-terminal amino acid (polypeptide-COOH) is modified toform an amide (polypeptide-CONH₂). The amide may be formed bypost-translational C-terminal amidation. The amino acid to be modifiedcan be followed by a glycine, which provides the amide group. Theprocess of post-translational amidation of a polypeptide derived from aprecursor proprotein is well characterized and involves three enzymaticsteps (Cuttitta, The Anatomical Record, 236:87-93, 1993). Step oneinvolves endoproteolytic cleavage at a pair of basic amino acids nearthe carboxy terminus of the protein. Step two involvescarboxypeptidase-mediated removal of basic residues. Step three is theamidation reaction, which involves oxidation of a terminal glycine toform the amide of the neighboring carboxy terminal amino acid. Glycineis the only known amino acid to function as an amide donor for itsneighboring amino acid. Although the free acid and amidated forms of apolypeptide are difficult to distinguish structurally, the amide can be100-1000 times more biologically active than the free acid form of thepolypeptide (Cuttitta, The Anatomical Record, 236:87-93, 1993).C-terminal amidation is essential to the biological activity of manypolypeptides.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Collagen: Proteins that are found in the form of elongated fibrils inmammals that are mostly found in fibrous tissues such as tendon,ligament and skin, and is also abundant in cornea, cartilage, bone,blood vessels, the gut, and intervertebral disc. The tropocollagen or“collagen molecule” is a subunit of larger collagen aggregates such asfibrils. It is approximately 300 nm long and 1.5 nm in diameter, made upof three polypeptide strands (called alpha chains), each possessing theconformation of a left-handed helix. In type I collagen, eachtriple-helix associates into a right-handed super-super-coil that isreferred to as the collagen microfibril. Endostatin is the first 183amino acids of collagen.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease an activityof a C-terminal endostatin polypeptide, such as the ability of thepolypeptide to inhibit fibrosis. Specific, non-limiting examples ofconservative substitutions include the following:

Original Residue Conservative Substitution(s) Ala Ser Arg Lys Asn Gln,His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile;Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser TrpTyr Tyr Trp; Phe Val Ile; Leu

The term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide. Non-conservative substitutions are thosethat reduce an activity, such as the ability of a protein to inhibitfibrosis.

Consists Essentially Of/Consists Of: With regard to a polypeptide, apolypeptide that consists essentially of a specified amino acid sequencedoes not include any additional amino acid residues. However, thepolypeptide can include additional non-peptide components, such aslabels (for example, fluorescent, radioactive, or solid particlelabels), sugars or lipids, that do not materially affect the basic andnovel characteristics of the polypeptide. A polypeptide that consists ofa specified amino acid sequence does not include any additional aminoacid residues, nor does it include additional biological components,such as nucleic acids lipids, sugars, nor does it include labels. Apolypeptide that consists or consists essential of a specified aminoacid sequence can be glycosylated or have an amide modification. Withregard to a polynucleotide, a polynucleotide that consists essentiallyof a specified nucleic acid sequence does not include any additionalnucleic acid residues. However, the polynucleotide can includeadditional non-nucleic acid components, such as labels (for example,fluorescent, radioactive, or solid particle labels) or polypeptides,that do not materially affect the basic and novel characteristic(s)” ofthe polynucleotides. A polynucleotide that consists of a specifiednucleic acid sequence does not include any additional nucleic acidresidues, nor does it include additional biological components orlabels.

Degenerate variant: A polynucleotide encoding a C-terminal endostatinpolypeptide that includes a sequence that is degenerate as a result ofthe genetic code. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are included in this disclosure as long as the amino acidsequence of the C-terminal endostatin polypeptide encoded by thenucleotide sequence is unchanged.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus, expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see e.g., Bitter etal., Meth Enzymol 153:516-544, 1987). For example, when cloning inbacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can beused. In some embodiments, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (such as themetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques can also be used to provide fortranscription of the nucleic acid sequences.

Endostatin: A 183 amino acid proteolytic cleavage fragment correspondingto the C-terminus of collagen XVIII. C-terminal polypeptides ofendostatin include consecutive amino acids from the C-terminal region,which is from amino acid 93 to amino acid 183. Exemplary humanendostatin polypeptides are set forth in SEQ ID NO:2 and SEQ ID NO:13.

Fibrosis: The formation or development of excess fibrous connectivetissue in an organ or tissue as a reparative or reactive process, asopposed to a formation of fibrous tissue as a normal constituent of anorgan or tissue. Skin and lungs are susceptible to fibrosis. Exemplaryfibrotic conditions are scleroderma idiopathic pulmonary fibrosis,morphea, fibrosis as a result of Graft-Versus-Host Disease (GVHD),keloid and hypertrophic scar, and subepithelial fibrosis, endomyocardialfibrosis, uterine fibrosis, myelofibrosis, retroperitoneal fibrosis,nephrogenic systemic fibrosis, scarring after surgery, asthma,cirrhosis/liver fibrosis, aberrant wound healing, glomerulonephritis,and multifocal fibrosclerosis.

Heterologous: Originating from separate genetic sources or species. Apolypeptide that is heterologous to endostatin originates from a nucleicacid that does not encode endostatin. In specific, non-limitingexamples, a polypeptide containing a C-terminal endostatin polypeptideand a heterologous amino acid sequence includes an Ig (such as IgG1)β-galactosidase, a maltose binding protein, and albumin, hepatitis Bsurface antigen, or an immunoglobulin amino acid sequence. Generally, anantibody that specifically binds to a protein of interest such asendostatin will not specifically bind to a heterologous protein.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The cell can bemammalian, such as a human cell. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used.

Idiopathic Pulmonary Fibrosis: A condition also known as cryptogenicfibrosing alveolitis (CFA) that is a chronic, progressive form of lungdisease characterized by fibrosis of the supporting framework(interstitium) of the lungs. By definition, the term is used only whenthe cause of the pulmonary fibrosis is unknown (“idiopathic”). When lungtissue from patients with IPF is examined under a microscope by apathologist, it shows a characteristic set of histologic/pathologicfeatures known as usual interstitial pneumonia (UIP). UIP ischaracterized by progressive scarring of both lung that involves thesupporting framework (interstitium) of the lung.

Inhibiting or treating a disease: Inhibiting a disease, such asfibrosis, refers to inhibiting the full development of a disease. Inseveral examples, inhibiting a disease refers to lessening symptoms of afibrosis, such as the formation of scar tissue or an increase in rangeof motion or a decrease in pain. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition related to the disease, such as the fibrosis.

Isolated: An “isolated” biological component (such as a nucleic acid orprotein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, e.g., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

Keloid or keloidal scar: A type of scar, which depending on itsmaturity, is composed of mainly either type III (early) or type I (late)collagen. It is a result of an overgrowth of granulation tissue(collagen type 3) at the site of a healed skin injury which is thenslowly replaced by collagen type 1. Keloids are firm, rubbery lesions orshiny, fibrous nodules, and can vary from pink to flesh-colored or redto dark brown in color. A keloid scar is benign, non-contagious, andusually accompanied by severe itchiness, sharp pains, and changes intexture. In severe cases, it can affect movement of skin. Keloids aredifferent than hypertrophic scars, which are raised scars that do notgrow beyond the boundaries of the original wound.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, enzymatic linkages, and radioactive isotopes.

Linker sequence: A linker sequence is an amino acid sequence thatcovalently links two polypeptide domains. Linker sequences can beincluded in the between the C-terminal endostatin polypeptides disclosedherein to provide rotational freedom to the linked polypeptide domainsand thereby to promote proper domain folding and presentation to theMHC. By way of example, in a recombinant polypeptide containing twoC-terminal endostatin polypeptides, linker sequences can be providedbetween them, such as a poly peptide containing C-terminal endostatinpolypeptide-linker-C-terminal endostatin polypeptide. Linker sequences,which are generally between 2 and 25 amino acids in length, are wellknown in the art and include, but are not limited to, theglycine(4)-serine spacer (×3) described by Chaudhary et al., Nature339:394-397, 1989.

Lysyl oxidase (LOX): Lysyl oxidase is an extracellular copper enzymethat catalyzes formation of aldehydes from lysine residues in collagenand elastin precursors. These aldehydes are highly reactive, and undergospontaneous chemical reactions with other lysyl oxidase-derived aldehyderesidues, or with unmodified lysine residues. This results incross-linking collagen and elastin, which is essential for stabilizationof collagen fibrils and for the integrity and elasticity of matureelastin.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Matrix metalloproteinase-2: A 72 kDa type IV collagenase also known asgelatinase A. Proteins of the matrix metalloproteinase (MMP) family areinvolved in the breakdown of extracellular matrix in normalphysiological processes, such as embryonic development, reproduction,and tissue remodeling, as well as in disease processes, such asarthritis and metastasis. Most MMP's are secreted as inactiveproproteins which are activated when cleaved by extracellularproteinases. MMP-2 degrades type IV collagen, the major structuralcomponent of basement membranes. MMP-2 also degrades additionalsubstrates such as native and denatured collagen I and fibronectin (seethe clip.ubc.ca/archive/mmp_timp_folder/mmp_substrates.shtm website).

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a polypeptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence, such as a sequence that encodes a C-terminal endostatinpolypeptide. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Peptide Modifications: C-terminal endostatin polypeptides includesynthetic embodiments of polypeptides described herein. In addition,analogs (non-peptide organic molecules), derivatives (chemicallyfunctionalized polypeptide molecules obtained starting with thedisclosed polypeptide sequences) and variants (homologs) of theseproteins can be utilized in the methods described herein. Eachpolypeptide of this disclosure is comprised of a sequence of aminoacids, which may be either L- and/or D-amino acids, naturally occurringand otherwise.

Peptides can be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpolypeptides, and optionally having other desirable properties. Forexample, carboxylic acid groups of the protein, whethercarboxyl-terminal or side chain, can be provided in the form of a saltof a pharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ester, or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ areeach independently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the polypeptide,whether amino-terminal or side chain, can be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or can be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the polypeptide side chains may be converted toC₁-C₁₆ alkoxy or to a C₁-C₁₆ ester using well-recognized techniques.Phenyl and phenolic rings of the polypeptide side chains may besubstituted with one or more halogen atoms, such as fluorine, chlorine,bromine or iodine, or with C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acidsand esters thereof, or amides of such carboxylic acids. Methylene groupsof the polypeptide side chains can be extended to homologous C₂-C₄alkylenes. Thiols can be protected with any one of a number ofwell-recognized protecting groups, such as acetamide groups. Thoseskilled in the art will also recognize methods for introducing cyclicstructures into the polypeptides of this invention to select and provideconformational constraints to the structure that result in enhancedstability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe polypeptide backbone and component amino acid side chains, resultingin such peptido- and organomimetics of C-terminal endostatin polypeptidehaving measurable or enhanced ability to treat fibrosis. For computermodeling applications, a pharmacophore is an idealized three-dimensionaldefinition of the structural requirements for biological activity.Peptido- and organomimetics can be designed to fit each pharmacophorewith current computer modeling software (using computer assisted drugdesign or CADD). See Walters, “Computer-Assisted Modeling of Drugs,” inKlegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, InterpharmPress: Buffalo Grove, Ill., pp. 165-174 and Principles of Pharmacology,Munson (ed.) 1995, Ch. 102, for descriptions of techniques used in CADD.Also included are mimetics prepared using such techniques.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975),describes compositions and formulations suitable for pharmaceuticaldelivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually include injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

A “therapeutically effective amount” is a quantity of a composition toachieve a desired effect in a subject being treated. For instance, thiscan be the amount necessary to induce an immune response, inhibitfibrosis, reduce scar volume, or measurably alter outward symptoms ofthe fibrotic condition. When administered to a subject, a dosage willgenerally be used that will achieve target tissue concentrations (forexample, in skin cells or lung tissue) that has been shown to achieve anin vitro effect.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Peptide or Polypeptide: Any chain of amino acids, regardless of lengthor post-translational modification (e.g., glycosylation orphosphorylation). In some embodiments, the polypeptide is a C-terminalendostatin polypeptide. A polypeptide can be between 5 and 60 aminoacids in length. In some embodiments, a polypeptide is from about 10 toabout 55 amino acids in length. In yet another embodiment, a polypeptideis from about 20 to about 50 amino acids in length. In yet anotherembodiment, polypeptide is about 50 amino acids in length. With regardto polypeptides, the word “about” indicates integer amounts. Thus, inone example, a polypeptide “about” 50 amino acids in length is from 49to 51 amino acids in length. In some embodiments, a polypeptide can bein multimeric form. For example, a polypeptide can be a high molecularweight multimer that includes a plurality of endostatin fragments and/orfusions as described herein. A high molecular weight multimer can have amolecular weight greater than 500 kDa, for example, (e.g., 500 to 750kDa, 750 kDa to 1 mega-Dalton (MDa), 1 to 1.5 MDa, 1.5 to 2 MDa, 2 to2.5 MDa, or 2.5 to 3 MDa).

Post-translational modification: The modification of a newly formedprotein; may involve deletion of amino acids, chemical modification ofcertain amino acids (for example, amidation, acetylation,phosphorylation, glycosylation, formation of pyroglutamate,oxidation/reduction of sulfa group on a methionine, or addition ofsimilar small molecules) to certain amino acids.

Probes and primers: A probe includes an isolated nucleic acid attachedto a detectable label or reporter molecule. Primers are short nucleicacids, preferably DNA oligonucleotides, of about 15 nucleotides or morein length. Primers may be annealed to a complementary target DNA strandby nucleic acid hybridization to form a hybrid between the primer andthe target DNA strand, and then extended along the target DNA strand bya DNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, for example by polymerase chain reaction (PCR) orother nucleic-acid amplification methods known in the art. One of skillin the art will appreciate that the specificity of a particular probe orprimer increases with its length. Thus, for example, a primer containing20 consecutive nucleotides will anneal to a target with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers can be selectedthat contain about 20, 25, 30, 35, 40, 50 or more consecutivenucleotides.

Purified: The C-terminal endostatin polypeptides disclosed herein can bepurified (and/or synthesized) by any of the means known in the art (see,e.g., Guide to Protein Purification, ed. Deutscher, Meth. Enzymol. 185,Academic Press, San Diego, 1990; and Scopes, Protein Purification:Principles and Practice, Springer Verlag, New York, 1982). Substantialpurification denotes purification from other proteins or cellularcomponents. A substantially purified protein is at least about 60%, 70%,80%, 90%, 95%, 98% or 99% pure. Thus, in one specific, non-limitingexample, a substantially purified protein is 90% free of other proteinsor cellular components.

Thus, the term purified does not require absolute purity; rather, it isintended as a relative term. For example, a purified nucleic acid is onein which the nucleic acid is more enriched than the nucleic acid in itsnatural environment within a cell. In additional embodiments, a nucleicacid or cell preparation is purified such that the nucleic acid or cellrepresents at least about 60% (such as, but not limited to, 70%, 80%,90%, 95%, 98% or 99%) of the total nucleic acid or cell content of thepreparation, respectively.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids. e.g., by genetic engineering techniques.

Scleroderma: A chronic autoimmune disease characterized by fibrosis (orhardening), vascular alterations, and autoantibodies. There are twomajor forms, one is a systemic form that includes limited cutaneousscleroderma mainly affects the hands, arms and face, although pulmonaryhypertension is frequent. Diffuse cutaneous scleroderma (or systemicsclerosis) is rapidly progressing and affects a large area of the skinand one or more internal organs, frequently the kidneys, esophagus,heart and lungs. Systemic scleroderma in both of its forms can be fatal.The other form of scleroderma is a localized form that has two subtypes:morphea and linear scleroderma. The disclosed endostatin peptides can beused to treat any form of scleroderma.

Selectively hybridize: Hybridization under moderately or highlystringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (for example, GC v. ATcontent), and nucleic acid type (for example, RNA versus DNA) of thehybridizing regions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

A specific example of progressively higher stringency conditions is asfollows: 2×SSC/0.1% SDS at about room temperature (hybridizationconditions); 0.2×SSC/0.1% SDS at about room temperature (low stringencyconditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringencyconditions); and 0.1×SSC at about 68° C. (high stringency conditions).One of skill in the art can readily determine variations on theseconditions (e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Washing can be carried out using only one ofthese conditions, e.g., high stringency conditions, or each of theconditions can be used, e.g., for 10-15 minutes each, in the orderlisted above, repeating any or all of the steps listed. However, asmentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a C-terminal endostatin polypeptide will possessa relatively high degree of sequence identity when aligned usingstandard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Higgins and Sharp, Gene 73:237, 1988; Higginsand Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a C-terminal endostatin polypeptide aretypically characterized by possession of at least 75%, for example atleast 80%, sequence identity counted over the full length alignment withthe amino acid sequence of endostatin using the NCBI Blast 2.0, gappedblastp set to default parameters. For comparisons of amino acidsequences of greater than about 30 amino acids, the Blast 2 sequencesfunction is employed using the default BLOSUM62 matrix set to defaultparameters, (gap existence cost of 11, and a per residue gap cost of 1).When aligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and can possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are available at the NCBIwebsite on the interne. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Therapeutically effective amount: A quantity of compound, such as theC-terminal endostatin polypeptide sufficient to achieve a desired effectin a subject being treated. For instance, this can be the amountnecessary to treat or ameliorate fibrosis, such as skin or lungfibrosis, in a subject. In some embodiments, it is the amount necessaryto treat a subject by a measurable amount over a period of time, or tomeasurably inhibit progression of disease, in a subject. In otherembodiments, a therapeutically effective amount is the amount necessaryto prophylactically inhibit a disease.

An effective amount of a C-terminal endostatin polypeptide may beadministered in a single dose, or in several doses, for example daily,during a course of treatment. However, the effective amount will bedependent on the compound applied, the subject being treated, theseverity and type of the affliction, and the manner of administration ofthe compound.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergene and other genetic elements known in the art. Vectors includeplasmid vectors, including plasmids for expression in gram negative andgram positive bacterial cell. Exemplary vectors include those forexpression in E. coli and Salmonella. Vectors also include viralvectors, such as, but are not limited to, retrovirus, orthopox, avipox,fowlpox, capripox, suipox, adenoviral, herpes virus, alpha virus,baculovirus, Sindbis virus, vaccinia virus and poliovirus vectors.Vectors also include vectors for expression in yeast cells and insectcells.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or peptides areapproximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

C-Terminal Endostatin Polypeptide

C-terminal endostatin polypeptides and variants thereof are disclosedherein. The polypeptides can inhibit fibrosis present in fibroticconditions such as, without limitation, scleroderma. The polypeptidescontain a C-terminal amino acid sequence of an endostatin protein, butdo not include full length endostatin. The endostatin protein can be amammalian protein, such as from a human, a non-human primate, a canine,a feline, an equine, a bovine, an ovine, a sheep, or a rodent (e.g.,mouse or rat). An exemplary nucleotide sequence encoding humanendostatin (the amino acid sequence set forth as SEQ ID NO:2) is:

(SEQ ID NO: 1) ATGCACAGCC ACCGCGACTT CCAGCCGGTG CTCCACCTGGTTGCGCTCAA CAGCCCCCTG TCAGGCGGCA TGCGGGGCATCCGCGGGGCC GACTTCCAGT GCTTCCAGCA GGCGCGGGCC GTGGGGCTGG CGGGCACCTT CCGCGCCTTC CTGTCCTCGCGCCTGCAGGA CCTGTACAGC ATCGTGCGCC GTGCCGACCGCGCAGCCGTG CCCATCGTCA ACCTCAAGGA CGAGCTGCTG TTTCCCAGCT GGGAGGCTCT GTTCTCAGGC TCTGAGGGTCCGCTGAAGCC CGGGGCACGC ATCTTCTCCT TTAACGGCAAGGACGTCCTG ACCCACCCCA CCTGGCCCCA GAAGAGCGTG TGGCATGGCT CGGACCCCAA CGGGCGCAGG CTGACCGAGAGCTACTGTGA GACGTGGCGG ACGGAGGCTC CCTCGGCCACGGGCCAGGCC TACTCGCTGC TGGGGGGCAG GCTCCTGGGG CAGAGTGCCG CGAGCTGCCA TCACGCCTAC ATCGTGCTATGCATTGAGAA CAGCTTCATG ACTGCCTCCA AGTAG

See also GENBANK® Accession Nos. NM030582.3; NM130444.2; NM130445.2, allof which are incorporated herein by reference.

Another exemplary nucleotide sequence encoding a human endostatin (theamino acid sequence set forth as SEQ ID NO:13) is:

(SEQ ID NO: 12) CACAGCCACCGC GACTTCCAGC CGGTGCTCCACCTGGTTGCGCTCAACAGCC CCCTGTCAGG CGGCATGCGG GGCATCCGCGGGGCCGACTTCCAGTGCTTC CAGCAGGCGC GGGCCGTGGG GCTGGCGGGC ACCTTCCGCG CCTTCCTGTCCTCGCGCCTGCAGGACCTGT ACAGCATCGT GCGCCGTGCC GACCGCGCAGCCGTGCCCATCGTCAACCTC AAGGACGAGC TGCTGTTTCC CAGCTGGGAG GCTCTGTTCT CAGGCTCTGAGGGTCCGCTGAAGCCCGGGG CACGCATCTT CTCCTTTGAC GGCAAGGACGTCCTGAGGCACCCCACCTGG CCCCAGAAGA GCGTGTGGCA TGGCTCGGAC CCCAACGGGC GCAGGCTGACCGAGAGCTACTGTGAGACGT GGCGGACGGA GGCTCCCTCG GCCACGGGCCAGGCCTCCTCGCTGCTGGGG GGCAGGCTCC TGGGGCAGAG TGCCGCGAGC TGCCATCACG CCTACATCGTGCTCTGCATTGAGAACAGCT TCATGACTGC CTCCAAGTAG

The exemplary human endostatin polypeptide sequence encoded by SEQ IDNO:1 is:

(SEQ ID NO: 2) HSHRDFQPVL HLVALNSPLS GGMRGIRGAD FQCFQQARAVGLAGTFRAFL SSRLQDLYSI VRRADRAAVP IVNLKDELLFPSWEALFSGS EGPLKPGARI FSFNGKDVLT HPTWPQKSVW HGSDPNGRRL TESYCETWRT EAPSATGQAY SLLGGRLLGQ SAASCHHAYI VLCIENSFMTASK 

This protein is 183 amino acids in length, and is identical to GENBANK®Accession number AAF01310 except that it is lacking the initiatormethionine of AAF01310).

The exemplary human endostatin polypeptide sequence encoded by SEQ IDNO:12 is:

(SEQ ID NO: 13) HSHRDFQPVL HLVALHSPLS GGMRGIRGAD FQCFQQARAVGLAGTFRAFL SSRLQDLYSI VRRADRAAVP IVNLKDELLFPSWEALFSGS EGPLKPGARI FSFDGKDVLR HPTWPQKSVW HGSDPNGRRL TESYCETWRT EAPSATGQAS SLLGGRLLGQ SAASCHHAYI VLCIENSFMTASK

See also GENBANK® Accession No, CAB90482, which is incorporated hereinby reference.

SEQ ID NO:2 is identical to SEQ ID NO:13, with the exception of threeamino acid substitutions, indicated by underlining in SEQ ID NOS:2 and13 above.

An exemplary nucleotide sequence encoding mouse endostatin is:

(SEQ ID NO: 3) CATACTCATC AGGACTTTCA GCCAGTGCTC CACCTGGTGGCACTGAACAC CCCCCTGTCT GGAGGCATGC GTGGTATCCGTGGAGCAGAT TTCCAGTGCT TCCAGCAAGC CCGAGCCGTG GGGCTGTCGG GCACCTTCCG GGCTTTCCTG TCCTCTAGGCTGCAGGATCT CTATAGCATC GTGCGCCGTG CTGACCGGGGGTCTGTGCCC ATCGTCAACC TGAAGGACGA GGTGCTATCT CCCAGCTGGG ACTCCCTGTT TTCTGGCTCC CAGGGTCAACTGCAACCCGG GGCCCGCATC TTTTCTTTTG ACGGCAGAGATGTCCTGAGA CACCCAGCCT GGCCGCAGAA GAGCGTATGG CACGGCTCGG ACCCCAGTGG GCGGAGGCTG ATGGAGAGTTACTGTGAGAC ATGGCGAACT GAAACTACTG GGGCTACAGGTCAGGCCTCC TCCCTGCTGT CAGGCAGGCT CCTGGAACAG AAAGCTGCGA GCTGCCACAA CAGCTACATC GTCCTGTGCATTGAGAATAG CTTCATGACC TCTTTCTCCA AA 

The exemplary mouse endostatin polypeptide sequence encoded by SEQ IDNO:3 is:

(SEQ ID NO: 4) HTHQDFQPVL HLVALNTPLS GGMRGIRGAD FQCFQQARAVGLSGTFRAFL SSRLQDLYSI VRRADRGSVP IVNLKDEVLSPSWDSLFSGS QGQLQPGARI FSFDGRDVLR HPAWPQKSVW HGSDPSGRRL MESYCETWRT ETTGATGQAS SLLSGRLLEQ KAASCHNSYI VLCIENSFMT SFSK

This protein is identical to GENBANK® Accession number AAF69009.Endostatin nucleotide and amino acid sequences from other species alsoare publicly available.

In some embodiments, the C-terminal endostatin polypeptide containsabout 10 to about 60 consecutive amino acids of the C-terminal region ofan endostatin protein, but does not include a full length endostatinprotein, or the N-terminal region of an endostatin protein. The peptidecan include from about 10 to about 55 consecutive amino acids or fromabout 20 to about 54 consecutive amino acids of the C-terminal region ofan endostatin protein, such as about 53 consecutive amino acids of theC-terminal region of an endostatin protein (such as SEQ ID NO:2). Forexample, the peptide may include about 40, about 45, about, 46, about47, about 48, about 49, about 50, about 51, about 52 or about 53consecutive amino acids of the C-terminal region of an endostatinprotein, such as amino acids 93 to 183 of an endostatin sequence such asSEQ ID NO:2, SEQ ID NO:13, or SEQ ID NO:4. In the context of an aminoacid or nucleic acid sequence, “about” means within one residue (onemore or one less than the specified number).

The endostatin peptide can include 40, 45, 46, 47, 48, 49, 50, 51, 52,53, 40-45, 45-50, or 50-55 consecutive amino acids of the C-terminalregion of an endostatin protein. In some examples the peptide consistsof 40, 45, 46, 47, 48, 49, 50, 51, 52, 53 consecutive amino acids of theC-terminal region of an endostatin polypeptide such as, withoutlimitation, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:13. In someembodiments, the peptide includes or consists of at least 30 amino acidsof amino acids 133 to 180 of endostatin, or a variant thereof that hasanti-fibrotic activity.

The endostatin peptide may include, consist of, or consist essentiallyof about amino acid 120, 125, 130, 131, 132, 133, 134, or 135 to aboutamino acid 175, 180, 181, 182 or 183 of an endostatin protein, such asSEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:13. In some examples, the peptideincludes, consists of, or consists essentially of amino acid 120 to 183,125 to 183, 130 to 183, 131 to 183, 132 to 183, 134 to 183, 135 to 183,120 to 180, 125 to 180, 130 to 180, 131 to 180, 132 to 180, 133 to 180,134 to 180, or 135 to 180 of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:13.In specific examples, the peptide includes, consists of, or consistsessentially of amino acids 133-180 of SEQ ID NO:2, amino acids 133-180of SEQ ID NO:4, amino acids 133-180 of SEQ ID NO:13. In this context,“consists essentially of” means that a peptide does not includeadditional amino acid residues but can include additional components,such as a label.

Other endostatin peptide variants disclosed herein may comprise, consistof or consist essentially of an amino acid sequence that has at leastabout 70%, 80%, 90%, 95%, 98% or 99% identity or homology with aC-terminal endostatin polypeptide. C-terminal endostatin polypeptides donot include a full length endostatin protein or the N-terminal region ofan endostatin protein (such as amino acids 1-92 of SEQ ID NO:2).

In some non-limiting examples, C-terminal endostatin polypeptides caninclude substitutions, such as conservative amino acid substitutions, ina naturally occurring C-terminal endostatin polypeptide (see SEQ IDNO:2, 4, or 13) in at most about 1, 2, 3, 4, 5 substitutions would beexpected to retain anti-fibrotic activity. The C-terminal endostatinpolypeptide can include at most 1, at most 2, at most 3 or at most 4amino acid substitutions, such as conservative amino acid substitutions.

Polypeptides that are similar to the sequences described above maycontain substitutions, deletions or additions. The differences can be,for example, in regions of the polypeptide that are not significantlyconserved among different species. Such regions can be identified byaligning the amino acid sequences of endostatin proteins from variousanimal species. Thus, the endostatin peptide can include, consistessentially of, or consist of at least 40, at least 45, at least 46, atleast 47, at least 48, at least 50, at least 51, at least 52, or all ofthe amino acids set forth as amino acids 133-180 of SEQ ID NO:2, SEQ IDNO:4, or SEQ ID NO:13. Alternatively, the endostatin peptide can includeat most 1, 2, 3, 4, or 5 amino acid substitutions in one of thesesequences, provided the peptide has anti-fibrotic activity. The peptidecan be 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 40-45, 45-50, or 50-55amino acids in length. The peptide does not include the entire sequenceof endostatin, or the N-terminal region, such as of SEQ ID NO:2, SEQ IDNO:4, or SEQ ID NO:13. In additional embodiments, the peptide is at most40, 45, 46, 47, 48, 49, 50, 51, 52, or 53 amino acids in length, such aspeptide that is 40, 45, 46, 47, 48, 49, 50, 51, 52, or 53 amino acids inlength.

In some embodiments, the peptide is modified, such as to include aC-terminal amide. Any of the C-terminal endostatin polypeptidesdisclosed herein can include a C-terminal amide.

C-terminal endostatin polypeptides include three cysteine residues inthe amino acid 133-180 of endostatin. Without being bound by theory, itis believed that two of the three cysteine residues participate informing an intramolecular disulfide bridge which is important to thetertiary structure of the peptide. The third cysteine residue mayparticipate in intermolecular interactions. In some embodiments,C-terminal endostatin polypeptides can include a cysteine modification.The cysteine modification may be substituted with any amino acid. Insome embodiments, the cysteine modification can be a cysteine to alaninesubstitution. Without being bound by theory, it is believed thatsubstitution of a cysteine with an alanine (e.g., C67A) can enhancesolubility of a C-terminal endostatin polypeptide as described herein.An exemplary C-terminal endostatin polypeptide having a cysteine toalanine substitution is set forth in SEQ ID NO:14.

The following is an alignment of the human (amino acids 133-180), mouse,rat, and cow collagen XVIII endostatin amino acid sequences, and humancollagen XV:

SEQ  ID:  Human SYCETWRTE

TGQASSLL

GRLL

Q

A

SCH

YIVLCIENSFMT  SYCETWRTE   ATGQASSLL GRLL Q AASCH +YIVLCIENSFMT  MouseSYCETWRTE

ATGQASSLL

GRLL

Q

AASCH

YIVLCIENSFMT  Rat SYCETWRTE

TGQASSLL

GRLL

QKA

SCH

YIVLCIENSFMT  Cow SYCETWRT

ATGQASSLL

GRLL

Q

AA

CH

R

TVLCIENSFMT  HumXV NYCEAWRTADTAVTGLASPLSTGKILDQKAYSCANRLIVLCIENSFMT Amino acids 133-141, 145-153, 155-158, 162-166, and 169-180 areconserved between the endostatin sequences (second line above). For thefull human endostatin sequence, see SEQ ID NO:2; for the full mouseendostatin sequence, see SEQ ID NO:4; for the full rat endostatinsequence, see SEQ ID NO:9; and for the full cow endostatin sequence, seeSEQ ID NO:10. The full human collagen XV is provided as SEQ ID NO:11.

In some embodiments, the amino acids in the second line of the abovealignment show regions of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:13 thatcan be conserved to preserve anti-fibrotic activity of the polypeptide.In further embodiments, the underlined amino acids should be retained inorder to preserve anti-fibrotic activity of the polypeptide. Thus, insome embodiments, the C-terminal endostatin polypeptide includes aminoacids 133-141, 145-153, 155-158, 162-166 and 169-180 of SEQ ID NO:2, SEQID NO:4 or SEQ ID NO:13. In some embodiments, the A at position 145 isconserved.

The polypeptide referred to herein as E4 is amino acids 133-180 of humanendostatin (see amino acids 133-180 of SEQ ID NO:2) with a C-terminalamide. In some embodiments, a region that can be retained in the peptideto retain anti-fibrotic activity includes one or both potentialphosphorylation sites in the first seven amino acids of E4 that areconserved: SYCE and TWR (amino acids 1-4 and 5-7 of E4, respectively,see also amino acids 133-136 and 137-139 of SEQ ID NO:2 or SEQ IDNO:13). In some embodiments, regions that can be retained in the peptideto retain anti-fibrotic activity include one or both potentialmyristoylation sites: GQaySL and GQsaAS (amino acids 15-20 and 27-32 ofE4, respectively, see amino acids 147-152 and amino acids 159-164 of SEQID NO:2 or SEQ ID NO:13). Thus, in some embodiments, the C-terminalendostatin polypeptide includes zero, or at most 1, at most 2, at most3, at most 4, or at most 5 substitutions in amino acids 133-180 of SEQID NO:2, SEQ ID NO:4, or SEQ ID NO:13, wherein the substitutions are notwithin amino acids 133-141, 145-153, 155-158, 162-166, and 169-180 ofSEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:13, and includes a C-terminalamide.

In other embodiments, amino acids can be substituted that differ betweenthe human and mouse sequences without affecting anti-fibrotic activity.In other embodiments, the bolded and italicized amino acids shown abovein the human sequence are those amino acids that can be substitutedwhile preserving anti-fibrotic activity. For example, amino acids142-144, 154, 159-161, and 181-183 of the amino acid sequence can bealtered in the C-terminal endostatin polypeptide. These amino acids canbe substituted, for example, with those found in another species, asshown above (SEQ ID NOs: 9-10). For example, the C-terminal endostatinpolypeptide can include amino acids 133-180 of SEQ ID NO:2 or SEQ IDNO:13, wherein amino acids 142-144, 154, and amino acids 159-161 aresubstituted. This polypeptide can include a C-terminal amide.

Other amino acids that can be substituted, inserted or deleted at theseor other locations can be identified by mutagenesis studies coupled withbiological assays. The above alignment is provided only as a guideline.

Also encompassed herein are multimers of polypeptides containingC-terminal endostatin polypeptides (e.g., E3), including fusionpolypeptides as described below. In some embodiments, a multimer can bea dimer, trimer, or tetramer, each comprising a C-terminal endostatinfragment.

Also encompassed herein are C-terminal endostatin polypeptides that arefused to a heterologous peptide, such as a peptide that can be used fordetecting, purifying, stabilizing, or solubilizing the endostatinpolypeptide. These polypeptides do not include a full length endostatinprotein or an N-terminal region of an endostatin protein. In someembodiments, a C-terminal polypeptide can be linked to an immunoglobulin(Ig) constant heavy or light chain domain or portion thereof at itsN-terminus. For example, a polypeptide such as, without limitation,amino acids 133-180 of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:13 (e.g.,E4), or SEQ ID NO:14 may be linked to a CH1, CH2 and/or CH3 domain of aheavy chain. If the constant region is from a light chain, it can befrom a kappa or lambda light chain. If the constant region is from aheavy chain, it can be from an antibody of any one of the followingclasses of antibodies: IgG, IgA, IgE, IgD, and IgM. IgG can be an IgG1,IgG2, IgG3 or IgG4. The constant domain may be an Fc fragment. Theconstant domain can be from a mammalian antibody, such as a humanantibody. Soluble receptor-IgG fusion proteins are common immunologicalreagents and methods for their construction are known in the art (see,for example, U.S. Pat. Nos. 5,225,538, 5,726,044; 5,707,632; 750,375,5,925,351, 6,406,697 and Bergers et al. Science 1999 284: 808-12). Inone example, the immunoglobulin is the constant part of the heavy chainof human IgG, particularly IgG1, where dimerization between two heavychains takes place at the hinge region. It is recognized that inclusionof the CH2 and CH3 domains of the Fc region as part of the fusionpolypeptide increases the in vivo circulation half-life of thepolypeptide comprising the Fc region, and that of the oligomer or dimercomprising the polypeptide.

An Fc portion of human IgG1 which includes the hinge region, and domainsCH2 and CH3 has the nucleotide sequence:

(SEQ ID NO: 5) GAG CCC AAA TCT TGT GAC AAA ACT CAC ACA TGC CCACCG TGC CCA GCA CCT GAA CTC CTG GGG GGA CCG TCAGTC TTC CTC TTC CCC CCA AAA CCC AAG GAC ACC CTCATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTGGTG GAC GTG AGC CAC GAA GAC CCT GAG GTC AAG TTCAAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCCAAG ACA AAG CCG CGG GAG GAG CAG TAC AAC AGC ACGTAC CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAGGAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTCTCC AAC AAA GCC CTC CCA GCC CCC ATC GAG AAA ACCATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAGGTG TAC ACC CTG CCC CCA TCC CGG GAT GAG CTG ACCAAG AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGCTTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGCAAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCTCCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TACAGC AAG CTC ACC GTG GAC AAG AGC AGG TGG CAG CAGGGG AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GCTCTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA TGA, which encodes a polypeptide having the amino acid sequence:

(SEQ ID NO: 6) Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys ProPro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro SerVal Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuMet Ile Ser Arg Thr Pro Glu Val Thr Cys Val ValVal Asp Val Ser His Glu Asp Pro Glu Val Lys PheAsn Trp Tyr Val Asp Gly Val Glu Val His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser ThrTyr Arg Val Val Ser Val Leu Thr Val Leu His GlnAsp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys ThrIle Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro GlnVal Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu ThrLys Asn Gln Val Ser Leu Thr Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu SerAsn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu TyrSer Lys Leu Thr Val Asp Lys Ser Arg Trp Gln GlnGly Asn Val Phe Ser Cys Ser Val Met His Glu AlaLeu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 

Constant Ig domains can also contain one or more mutations that reduceor eliminate one or more effector function, e.g., binding to Fcreceptors and complement activation (see, for example, Morrison, Annu.Rev. Immunol., 10, pp. 239-65 (1992); Duncan and Winter (1988) Nature332: 738-740; and Xu et al. (1994) J Biol. Chem. 269: 3469-3474). Forexample, mutations of amino acids corresponding to Leu 235 and Pro 331of human IgG1 to Glu and Ser respectively, are provided. Such constructsare further described in U.S. Pat. No. 6,656,728.

The C-terminal endostatin polypeptide can also be linked to a linkersequence with a thrombin cleavage site, such as between the C-terminalendostatin polypeptide and a heterologous polypeptide. An exemplarynucleotide sequence encoding such a site has the following nucleotidesequence: 5′ TCT AGA GGT GGT CTA GTG CCG CGC GGC AGC GGT TCC CCC GGG TTGCAG 3′ (SEQ ID NO:7), which encodes a polypeptide having the amino acidsequence: Ser Arg Gly Gly Leu Val Pro Arg Gly Ser Gly Ser Pro Gly LeuGln (SEQ ID NO:8). A C-terminal endostatin polypeptide can also be fusedto a signal sequence. For example, when prepared recombinantly, anucleic acid encoding the peptide can be linked at its 5′ end to asignal sequence, such that the peptide is secreted from the cell.

Peptides can be used as a substantially pure preparation, such aswherein at least about 90% of the peptides in the preparation are thedesired peptide. Compositions comprising at least about 50%, 60%, 70%,or 80% of the desired peptide may also be used. Peptides can bedenatured or non-denatured and may be aggregated or non-aggregated as aresult thereof.

Other C-terminal endostatin polypeptides that are encompassed herein arethose that include modified amino acids. Exemplary peptides arederivative peptides that may be one modified by glycosylation,pegylation, phosphorylation or any similar process that retains at leastone biological function of the peptide from which it was derived.Peptides may also comprise one or more non-naturally occurring aminoacids. For example, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into peptides.Non-classical amino acids include, but are not limited to, the D-isomersof the common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,gamma-Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyricacid, 3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,beta-alanine, fluoro-amino acids, designer amino acids such asbeta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl aminoacids, and amino acid analogs in general. In some embodiments,substitution with selenomethionine can be useful (e.g., for X-raydiffraction analysis). Further, the amino acid can be D (dextrorotary)or L (levorotary). In other specific embodiments, branched versions ofthe peptides listed herein are provided, such as by substituting one ormore amino acids within the sequence with an amino acid or amino acidanalog with a free side chain capable of forming a peptide bond with oneor more amino acids (and thus capable of forming a “branch”). Cyclicalpeptides are also contemplated.

Also included are peptide derivatives which are differentially modifiedduring or after synthesis, such as by benzylation, glycosylation,acetylation, phosphorylation, amidation, pegylation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. In specificembodiments, the peptides are acetylated at the N-terminus and/oramidated at the C-terminus.

In one example, the peptide includes a carboxy terminal amide. Onespecific non-limiting example of this type of C-terminal endostatinpolypeptide is E4 (see, for example, amino acids 133-180 of SEQ IDNO:13), which is described in detail in the examples section below. Thispeptide, or any of the C-terminal endostatin polypeptides disclosedherein can be amidated at the C-terminus.

Also provided are derivatives of C-terminal endostatin polypeptides,such as chemically modified peptides and peptidomimetics.Peptidomimetics are compounds based on, or derived from, peptides andproteins. Peptidomimetics can be obtained by structural modification ofknown peptide sequences using unnatural amino acids, conformationalrestraints, isosteric replacement, and the like. The subjectpeptidomimetics constitute the continuum of structural space betweenpeptides and non-peptide synthetic structures; peptidomimetics may beuseful, therefore, in delineating pharmacophores and in helping totranslate peptides into nonpeptide compounds with the activity of theparent peptides.

Mimetopes of the C-terminal endostatin polypeptides are included in thepresent disclosure. Such peptidomimetics can have such attributes asbeing non-hydrolyzable (e.g., increased stability against proteases orother physiological conditions which degrade the corresponding peptide),increased specificity and/or potency for stimulating celldifferentiation. For illustrative purposes, peptide analogs can begenerated using, for example, benzodiazepines (e.g., see Freidinger etal. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOMPublisher: Leiden, Netherlands, 1988), substituted gamma lactam rings(Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988, p 123), C-7 mimics (Huffmanet al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOMPublisher: Leiden, Netherlands, 1988, p. 105), keto-methylenepseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson etal. in Peptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), β-turndipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1: 1231), β-aminoalcohols (Gordonet al. (1985) Biochem Biophys Res Commun 126:419; and Dann et al. (1986)Biochem Biophys Res Commun 134:71), diaminoketones (Nataraj an et al.(1984) Biochem Biophys Res Commun 124:141), and methyleneamino-modified(Roark et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988, p 134). Also, see generally,Session III: Analytic and synthetic methods, in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988).

In addition to a variety of side-chain replacements which can be carriedout to generate peptidomimetics, the present disclosure specificallycontemplates the use of conformationally restrained mimics of peptidesecondary structure. Numerous surrogates have been developed for theamide bond of peptides. Frequently exploited surrogates for the amidebond include the following groups (i) trans-olefins, (ii) fluoroalkene,(iii) methyleneamino, (iv) phosphonamides, and (v) sulfonamides.Additionally, peptidomimetics based on more substantial modifications ofthe backbone of a peptide can be used. Peptidomimetics which fall inthis category include (i) retro-inverso analogs, and (ii) N-alkylglycine analogs (so-called peptoids). Furthermore, the methods ofcombinatorial chemistry can be used to produce peptidomimetics. Forexample, some embodiments of a so-called “peptide morphing” strategyfocus on the random generation of a library of peptide analogs thatcomprise a wide range of peptide bond substitutes. In an exemplaryembodiment, the peptidomimetic can be derived as a retro-inverso analogof the peptide. Such retro-inverso analogs can be made according to themethods known in the art, such as that described by the Sisto et al.U.S. Pat. No. 4,522,752. A retro-inverso analog can be generated asdescribed, for example in PCT Publication No. WO 00/01720. A mixedpeptide, such as one including some normal peptide linkages, can begenerated. As a general guide, sites which are most susceptible toproteolysis are typically altered, with less susceptible amide linkagesbeing optional for mimetic switching. The final product, orintermediates thereof, can be purified by HPLC.

In some embodiments, peptides can include at least one amino acid orevery amino acid that is a D stereoisomer. Other peptides can include atleast one amino acid that is reversed. The amino acid that is reversedmay be a D stereoisomer. Every amino acid of a peptide may be reversedand/or every amino acid can be a D stereoisomer. In another illustrativeembodiment, a peptidomimetic can be derived as a retro-enantio analog ofa peptide. Retro-enantio analogs such as this can be synthesized withcommercially available D-amino acids (or analogs thereof) and standardsolid- or solution-phase peptide-synthesis techniques, as described, forexample in PCT Publication No. WO 00/01720. The final product can bepurified by HPLC to yield the pure retro-enantio analog. In stillanother illustrative embodiment, trans-olefin derivatives can be madefor the subject peptide. Trans-olefin analogs can be synthesizedaccording to the method of Shue et al. (1987) Tetrahedron Letters28:3225 and as described in PCT Publication WO 00/01720. It is furtherpossible to couple pseudodipeptides synthesized by the above method toother pseudodipeptides, to make peptide analogs with several olefinicfunctionalities in place of amide functionalities. Still another classof peptidomimetic derivatives include the phosphonate derivatives. Thesynthesis of such phosphonate derivatives can be adapted from knownsynthesis schemes (see, for example, Loots et al. in Peptides: Chemistryand Biology, (Escom Science Publishers, Leiden, 1988, p. 118)); Petrilloet al. in Peptides: Structure and Function (Proceedings of the 9thAmerican Peptide Symposium, Pierce Chemical Co. Rockland, Ill., 1985).

Many other peptidomimetic structures are known in the art and can bereadily adapted for use in the subject peptidomimetics. For example, apeptidomimetic may incorporate the 1-azabicyclo[4.3.0]nonane surrogate(see Kim et al. (1997) J. Org. Chem. 62:2847), or an N-acyl piperazicacid (see Xi et al. (1998) J. Am. Chem. Soc. 120:80), or a 2-substitutedpiperazine moiety as a constrained amino acid analogue (see Williams etal. (1996) J. Med. Chem. 39:1345-1348). In still other embodiments,certain amino acid residues can be replaced with aryl and bi-arylmoieties, such as monocyclic or bicyclic aromatic or heteroaromaticnucleus, or a biaromatic, aromatic-heteroaromatic, or biheteroaromaticnucleus. The subject peptidomimetics can be optimized such as bycombinatorial synthesis techniques combined with high throughputscreening. Moreover, other examples of mimetopes include, but are notlimited to, protein-based compounds, carbohydrate-based compounds,lipid-based compounds, nucleic acid-based compounds, natural organiccompounds, synthetically derived organic compounds, anti-idiotypicantibodies and/or catalytic antibodies, or fragments thereof. A mimetopecan be obtained by, for example, screening libraries of natural andsynthetic compounds for compounds capable of inhibiting fibrosis. Amimetope can also be obtained, for example, from libraries of naturaland synthetic compounds, in particular, chemical or combinatoriallibraries (e.g., libraries of compounds that differ in sequence or sizebut that have the same building blocks). A mimetope can also be obtainedby, for example, rational drug design. In a rational drug designprocedure, the three-dimensional structure of a compound of the presentinvention can be analyzed by, for example, nuclear magnetic resonance(NMR) or x-ray crystallography. The three-dimensional structure can thenbe used to predict structures of potential mimetopes by, for example,computer modelling. The predicted mimetope structures can then beproduced by, for example, chemical synthesis, recombinant DNAtechnology, or by isolating a mimetope from a natural source (forexample, plants, animals, bacteria and fungi).

All of the C-terminal endostatin polypeptides of use in the disclosedmethods have anti-fibrotic activity. For example, they can reduce orinhibit fibrosis by a factor of at least about 50%, 60%, 70% 80%, 90%,or 2 fold, 5 fold, 10 fold, 30 fold or 100 fold, as compared to acontrol, such as in an assay described herein.

The C-terminal endostatin polypeptides (including amidated forms of thepeptides) can be readily synthesized by automated solid phase procedureswell known in the art. Techniques and procedures for solid phasesynthesis are described in Solid Phase Peptide Synthesis: A PracticalApproach, by E. Atherton and R. C. Sheppard, published by IRL, OxfordUniversity Press, 1989. Alternatively, these peptides may be prepared byway of segment condensation, as described, for example, in Liu et al.,Tetrahedron Lett. 37:933-936, 1996; Baca et al., J. Am. Chem. Soc.117:1881-1887, 1995; Tam et al., Int. J. Peptide Protein Res.45:209-216, 1995; Schnolzer and Kent, Science 256:221-225, 1992; Liu andTam, J. Am. Chem. Soc. 116:4149-4153, 1994; Liu and Tam, Proc. Natl.Acad. Sci. USA 91:6584-6588, 1994; and Yamashiro and Li, Int. J. PeptideProtein Res. 31:322-334, 1988). Other methods useful for synthesizingpeptides of the present disclosure are described in Nakagawa et al., J.Am. Chem. Soc. 107:7087-7092, 1985. Peptides of the disclosure can alsobe readily purchased from commercial suppliers of synthetic peptides.Such suppliers include, for example, Advanced ChemTech (Louisville,Ky.), Applied Biosystems (Foster City, Calif.), Anaspec (San Jose,Calif.), and Cell Essentials (Boston, Mass.).

Polynucleotides Encoding the C-Terminal Endostatin Polypeptides and HostCells

Polynucleotides encoding the C-terminal endostatin polypeptidesdisclosed herein are also provided. These polynucleotides include DNA,cDNA and RNA sequences which encode the peptide of interest. Silentmutations in the coding sequence result from the degeneracy (i.e.,redundancy) of the genetic code, whereby more than one codon can encodethe same amino acid residue. Thus, for example, leucine can be encodedby CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC,TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; asparticacid can be encoded by GAT or GAC; cysteine can be encoded by TGT orTGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can beencoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; andisoleucine can be encoded by ATT, ATC, or ATA. Tables showing thestandard genetic code can be found in various sources (see, for example,Stryer, 1988, Biochemistry, 3.sup.rd Edition, W. H. 5 Freeman and Co.,NY).

A nucleic acid encoding a C-terminal endostatin polypeptide can becloned or amplified by in vitro methods, such as the polymerase chainreaction (PCR), the ligase chain reaction (LCR), the transcription-basedamplification system (TAS), the self-sustained sequence replicationsystem (3SR) and the Qβ replicase amplification system (QB). Forexample, a polynucleotide encoding the protein can be isolated bypolymerase chain reaction of cDNA using primers based on the DNAsequence of the molecule. A wide variety of cloning and in vitroamplification methodologies are well known to persons skilled in theart. PCR methods are described in, for example, U.S. Pat. No. 4,683,195;Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; andErlich, ed., PCR Technology, (Stockton Press, NY, 1989). Polynucleotidesalso can be isolated by screening genomic or cDNA libraries with probesselected from the sequences of the desired polynucleotide understringent hybridization conditions.

The polynucleotides encoding a C-terminal endostatin polypeptide includea recombinant DNA which is incorporated into a vector in an autonomouslyreplicating plasmid or virus or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (such as a cDNA)independent of other sequences. The nucleotides can be ribonucleotides,deoxyribonucleotides, or modified forms of either nucleotide. The termincludes single and double forms of DNA.

In some embodiments, vectors are used for expression in yeast such as S.cerevisiae or Kluyveromyces lactis. Several promoters are known to be ofuse in yeast expression systems such as the constitutive promotersplasma membrane H⁺-ATPase (PMA1), glyceraldehyde-3-phosphatedehydrogenase (GPD), phosphoglycerate kinase-1 (PGK1), alcoholdehydrogenase-1 (ADH1), and pleiotropic drug-resistant pump (PDR5). Inaddition, many inducible promoters are of use, such as GAL1-10 (inducedby galactose), PHOS (induced by low extracellular inorganic phosphate),and tandem heat shock HSE elements (induced by temperature elevation to37° C.). Promoters that direct variable expression in response to atitratable inducer include the methionine-responsive MET3 and MET25promoters and copper-dependent CUP1 promoters. Any of these promotersmay be cloned into multicopy (2μ) or single copy (CEN) plasmids to givean additional level of control in expression level. The plasmids caninclude nutritional markers (such as URA3, ADE3, HIS1, and others) forselection in yeast and antibiotic resistance (AMP) for propagation inbacteria. Plasmids for expression on K lactis are known, such as pKLAC1.Thus, in one example, after amplification in bacteria, plasmids can beintroduced into the corresponding yeast auxotrophs by methods similar tobacterial transformation. The polynucleotides can also be designed toexpress in insect cells.

The C-terminal endostatin polypeptides can be expressed in a variety ofyeast strains. For example, seven pleiotropic drug-resistanttransporters, YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11, and PDR15, togetherwith their activating transcription factors, PDR1 and PDR3, have beensimultaneously deleted in yeast host cells, rendering the resultantstrain sensitive to drugs. Yeast strains with altered lipid compositionof the plasma membrane, such as the erg6 mutant defective in ergosterolbiosynthesis, can also be utilized. Proteins that are highly sensitiveto proteolysis can be expressed in a yeast lacking the master vacuolarendopeptidase Pep4, which controls the activation of other vacuolarhydrolases. Heterologous expression in strains carryingtemperature-sensitive (ts) alleles of genes can be employed if thecorresponding null mutant is inviable.

Viral vectors can also be prepared encoding the C-terminal endostatinpolypeptides disclosed herein. A number of viral vectors have beenconstructed, including polyoma, SV40 (Madzak et al., 1992, J. Gen.Virol., 73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol.Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629;Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992,Proc. Natl. Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell,68:143-155; Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239;Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256),vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499),adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol.158:91-123; On et al., 1990, Gene, 89:279-282), herpes viruses includingHSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol.,158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al.,1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol.,1:337-371; Fresse et al., 1990, Biochem. Pharmacol. 40:2189-2199),Sindbis viruses (Herweijer et al., 1995, Human Gene Therapy 6:1161-1167;U.S. Pat. Nos. 5,091,309 and 5,217,879), alphaviruses (Schlesinger,1993, Trends Biotechnol. 11:18-22; Frolov et al., 1996, Proc. Natl.Acad. Sci. USA 93:11371-11377) and retroviruses of avian (Brandyopadhyayet al., 1984, Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J.Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol.Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437;Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J.Virol., 54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739).Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors are also known in the art, and may be obtained fromcommercial sources (such as PharMingen, San Diego, Calif.; ProteinSciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).

Thus, in some embodiments, the polynucleotide encoding a C-terminalendostatin polypeptide is included in a viral vector. Suitable vectorsinclude retrovirus vectors, orthopox vectors, avipox vectors, fowlpoxvectors, capripox vectors, suipox vectors, adenoviral vectors, herpesvirus vectors, alpha virus vectors, baculovirus vectors, Sindbis virusvectors, vaccinia virus vectors and poliovirus vectors. Specificexemplary vectors are poxvirus vectors such as vaccinia virus, fowlpoxvirus and a highly attenuated vaccinia virus (MVA), adenovirus,baculovirus and the like.

Pox viruses of use include orthopox, suipox, avipox, and capripox virus.Orthopox include vaccinia, ectromelia, and raccoon pox. One example ofan orthopox of use is vaccinia. Avipox includes fowlpox, canary pox andpigeon pox. Capripox include goatpox and sheeppox. In one example, thesuipox is swinepox. Examples of pox viral vectors for expression asdescribed for example, in U.S. Pat. No. 6,165,460, which is incorporatedherein by reference. Other viral vectors that can be used include otherDNA viruses such as herpes virus and adenoviruses, and RNA viruses suchas retroviruses and polio.

Suitable vectors are disclosed, for example, in U.S. Pat. No. 6,998,252,which is incorporated herein by reference. In one example, a recombinantpoxvirus, such as a recombinant vaccinia virus is synthetically modifiedby insertion of a chimeric gene containing vaccinia regulatory sequencesor DNA sequences functionally equivalent thereto flanking DNA sequenceswhich in nature are not contiguous with the flanking vaccinia regulatoryDNA sequences that encode a C-terminal endostatin polypeptide. Therecombinant virus containing such a chimeric gene is effective atexpressing the C-terminal endostatin polypeptide. In one example, thevaccine viral vector comprises (A) a segment comprised of (i) a firstDNA sequence encoding a C-terminal endostatin polypeptide and (ii) apoxvirus promoter, wherein the poxvirus promoter is adjacent to andexerts transcriptional control over the DNA sequence encoding aC-terminal endostatin polypeptide; and, flanking said segment, (B) DNAfrom a nonessential region of a poxvirus genome. The viral vector canencode a selectable marker. In one example, the poxvirus includes, forexample, a thymidine kinase gene (see U.S. Pat. No. 6,998,252, which isincorporated herein by reference).

Poxviral vectors that encode a C-terminal endostatin polypeptide includeat least one expression control element operationally linked to thenucleic acid sequence encoding the C-terminal endostatin polypeptide.The expression control elements are inserted in the poxviral vector tocontrol and regulate the expression of the nucleic acid sequence.Examples of expression control elements of use in these vectorsincludes, but is not limited to, lac system, operator and promoterregions of phage lambda, yeast promoters and promoters derived frompolyoma, adenovirus, retrovirus or SV40. Additional operational elementsinclude, but are not limited to, leader sequence, termination codons,polyadenylation signals and any other sequences necessary for theappropriate transcription and subsequent translation of the nucleic acidsequence encoding the C-terminal endostatin polypeptide in the hostsystem. The expression vector can contain additional elements necessaryfor the transfer and subsequent replication of the expression vectorcontaining the nucleic acid sequence in the host system. Examples ofsuch elements include, but are not limited to, origins of replicationand selectable markers. It will further be understood by one skilled inthe art that such vectors are easily constructed using conventionalmethods (Ausubel et al., (1987) in Current Protocols in MolecularBiology, John Wiley and Sons, New York, N.Y.) and are commerciallyavailable.

Basic techniques for preparing recombinant DNA viruses containing aheterologous DNA sequence encoding the C-terminal endostatinpolypeptide, are known in the art. Such techniques involve, for example,homologous recombination between the viral DNA sequences flanking theDNA sequence in a donor plasmid and homologous sequences present in theparental virus (Mackett et al., 1982, Proc. Natl. Acad. Sci. USA79:7415-7419). In particular, recombinant viral vectors such as apoxviral vector can be used in delivering the gene. The vector can beconstructed for example by steps known in the art, such as stepsanalogous to the methods for creating synthetic recombinants of thefowlpox virus described in U.S. Pat. No. 5,093,258, incorporated hereinby reference. Other techniques include using a unique restrictionendonuclease site that is naturally present or artificially inserted inthe parental viral vector to insert the heterologous DNA.

DNA sequences encoding a C-terminal endostatin polypeptide can beexpressed in vitro by DNA transfer into a suitable host cell. The cellmay be prokaryotic or eukaryotic. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. Methods of stable transfer, meaning that the foreignDNA is continuously maintained in the host, are known in the art.

As noted above, a polynucleotide sequence encoding a C-terminalendostatin polypeptide can be operatively linked to expression controlsequences. An expression control sequence operatively linked to a codingsequence is ligated such that expression of the coding sequence isachieved under conditions compatible with the expression controlsequences. The expression control sequences include, but are not limitedto, appropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons.

Hosts cells can include microbial, yeast, insect and mammalian hostcells.

Methods of expressing DNA sequences having eukaryotic or viral sequencesin prokaryotes are well known in the art. Non-limiting examples ofsuitable host cells include bacteria, archea, insect, fungi (forexample, yeast), plant, and animal cells (for example, mammalian cells,such as human). Exemplary cells of use include Escherichia coli,Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9cells, C129 cells, 293 cells, Neurospora, and immortalized mammalianmyeloid and lymphoid cell lines. Techniques for the propagation ofmammalian cells in culture are well-known (see, Jakoby and Pastan (eds),1979, Cell Culture. Methods in Enzymology, volume 58, Academic Press,Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly usedmammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38,BHK, and COS cell lines, although cell lines may be used, such as cellsdesigned to provide higher expression desirable glycosylation patterns,or other features. As discussed above, techniques for the transformationof yeast cells, such as polyethylene glycol transformation, protoplasttransformation and gene guns are also known in the art (see Gietz andWoods, Meth Enzymol 350: 87-96, 2002).

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as, but not limited to, E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method using procedures well known in the art.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding a C-terminalendostatin polypeptide, and a second foreign DNA molecule encoding aselectable phenotype, such as the herpes simplex thymidine kinase gene.Another method is to use a eukaryotic viral vector, such as simian virus40 (SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

The endostatin fragments and variants also can be produced in plants.For example, polypeptides can be expressed in plants using theIBIOLAUNCH™ gene expression platform (iBio, Inc., Newark, Del.) asdescribed in, for example, U.S. Pat. No. 7,491,509 (which isincorporated herein by reference in its entirety). The IBIOLAUNCH™platform can be used to produce high levels of target proteins innon-transgenic plants. This platform can have benefits over methodsutilizing animal cells, or microbes, and over systems that requiretransgenic plants.

To use this system, a desired gene is cloned into an IBIOLAUNCH™ vector,which is introduced into the leaves of plants (e.g., by automated vacuuminfiltration). The vector is allowed to spread to cells in the stems andleaves, where the desired protein is expressed at extremely high levelsover the next 4-7 days. The green plant material is then harvested andthe protein product purified. The entire IBIOLAUNCH™ gene expressionprocess can be repeated for a different protein with a new plant crop inthe same facility, making this technology the most flexible and fastestway to produce protein drugs and vaccines.

A plant expression vector system can include one or more viral vectorcomponents. A wide variety of viruses are known that infect variousplant species, and can be employed for polynucleotide expression.Families of viruses that infect plants include, without limitation,Tobamoviridae, Caulimoviridae (dsDNA), Geminiviridae (ssDNA), Reoviridaeand Partitiviridae (dsRNA), and Rhabdoviridae, Bunyaviridae,Bromoviridae, and Comoviridae (ssRNA). Additional information can befound, for example, in “The Classification and Nomenclature of Viruses,Sixth Report of the International Committee on Taxonomy of Viruses” (Ed.Murphy et al.), Springer Verlag: New York, 1995, the entire contents ofwhich are incorporated herein by reference (see also, Grierson et al.,Plant Molecular Biology, Blackie, London, pp. 126-146, 1984; Gluzman etal., Communications in Molecular Biology: Viral Vectors, Cold SpringHarbor Laboratory, NY, pp. 172-189, 1988; and Mathew, Plant VirusesOnline.

In order to successfully establish either a local (intraleaf) orsystemic infection a virus must be able to replicate. Many virusescontain genes encoding one or more proteins that participate in thereplication process (referred to herein as replication proteins orreplicase proteins). For example, many RNA plant viruses encode an RNApolymerase. Additional proteins also may be required, such as helicaseor methyltransferase protein(s). The viral genome may contain varioussequence components in addition to functional genes encoding replicationproteins, which are also required for or facilitate replication.

Any virus that infects plants can be used to prepare a viral vector orvector system in accordance with the methods described herein. ssRNAviruses can be particularly useful, especially those with a (+)-strandedgenome. Techniques and reagents for manipulating the genetic materialpresent in such viruses include those that are well known in the art.Typically, for example, a DNA copy of the viral genome is prepared andcloned into a microbial vector (e.g., a bacterial vector). Certain ssDNAviruses, including geminiviruses, are particularly useful. It will beappreciated that in general the vectors and viral genomes may exist inRNA or DNA form. In addition, where reference is made to a feature suchas a genome or portion thereof of an RNA virus, which is present withina DNA vector, it is to be understood that the feature is present as theDNA copy of the RNA form.

Viruses of a number of different types may be used. Suitable virusesinclude, for example, members of the Bromoviridae (e.g., bromoviruses,alfamoviruses, ilarviruses) and Tobamoviridae. Useful virus speciesinclude, for example, Alfalfa Mosaic Virus (AlMV), Apple Chlorotic LeafSpot Virus, Apple Stem Grooving Virus, Barley Stripe Mosaic Virus,Barley Yellow Dwarf Virus, Beet Yellow Virus, Broad Bean Mottle Virus,Broad Bean Wilt Virus, Brome Mosaic Virus (BMV), Carnation Latent Virus,Carnation Mottle Virus, Carnation Ringspot Virus, Carrot Mottle Virus,Cassava Latent Virus (CLV), Cowpea Chlorotic Mottle Virus, Cowpea MosaicVirus (CPMV), Cucumber Green Mottle Mosaic Virus, Cucumber Mosaic Virus,Lettuce Infectious Yellow Virus, Maize Chlorotic Mottle Virus, MaizeRayado Fino Virus, Maize Streak Virus (MSV), Parsnip Yellow Fleck Virus,Pea Enation Mosaic Virus, Potato Virus X, Potato Virus Y, RaspberryBushy Dwarf Virus, Rice Necrosis Virus (RNV), Rice Stripe Virus, RiceTungro Spherical Virus, Ryegrass Mosaic Virus, Soil-borne Wheat MosaicVirus, Southern Bean Mosaic Virus, Tobacco Etch Virus (TEV), TobaccoMosaic Virus (TMV), Tobacco Necrosis Virus, Tobacco Rattle Virus,Tobacco Ring Spot Virus, Tomato Bushy Stunt Virus, Tomato Golden MosaicVirus (TGMV), and Turnip Yellow Mosaic Virus (TYMV).

Elements of these plant viruses are genetically engineered according toknown techniques (see, for example, (see, for example, Sambrook et al.,Molecular Cloning, 2^(nd) Edition, Cold Spring Harbor Press, NY, 1989;Clover et al., Molecular Cloning, IRL Press, Oxford, 1985; Dason et al.,Virology, 172:285-292, 1989; Takamatsu et al., EMBO J. 6:307-311, 1987;French et al., Science 231: 1294-1297, 1986; Takamatsu et al., FEBSLett. 269:73-76, 1990; Yusibov and Loesch-Fries, Virology, 208(1):405-7, 1995; and Spitsin et al., Proc. Natl. Acad. Sci. USA, 96(5):2549-53, 1999) to generate viral vectors for use in plant production ofpolypeptides of interest, including the endostatin polypeptides providedherein. At least two vectors are employed, one or both of which areincapable of systemic infection, but which together provide allfunctions needed to support systemic infection of at least one of thevectors and allow expression of a polynucleotide of interest throughoutthe plant. Thus, the viral components can complement each other in transto provide systemic infection capability.

In particular, a producer vector is prepared. This vector includes apolynucleotide of interest (e.g., a polynucleotide encoding anendostatin polypeptide) under control of regulatory sequences thatdirect expression in the relevant plant host. In some embodiments, thepolynucleotide is placed under control of a viral promoter, for examplethe CP promoter. For instance, it can be desirable to replace thenatural viral CP gene with the polynucleotide of interest. The producervector lacks one or more components required for systemic movement. Forexample, the producer vector may not contain sequences sufficient forexpression of functional CP (e.g., a CP gene), but may include a geneencoding a cell-to-cell movement protein. The producer vector maycontain one or more sequence elements (e.g., an origin of assembly) thatmay be required in cis to facilitate spread of the virus when present incis. For example, the producer vector may contain an origin of assemblythat is needed for or facilitates activity of a CP, either from the sametype of virus as the producer virus or from another virus. Such sequenceelements may comprise a recognition site for a CP. In other embodiments,the producer vector may lack sequences sufficient for expression offunctional MP and/or replicase proteins. In these embodiments, theproducer vector may or may not lack sequences sufficient for expressionof functional CP.

A carrier vector also is prepared. This vector complements the producervector, such that it provides components needed for systemic infectionthat are missing in the producer vector. For example, certain carriervectors include a functional coat protein encoding component. Thesecarrier vectors are suitable for complementing a producer vector thatlacks a functional coat protein encoding component. The carrier vectormay lack at least one viral component (e.g., a gene encoding a replicaseor movement protein) required for successful systemic infection of aplant, provided that such component is not also absent in the producervector. The carrier vector may include a polynucleotide of interest(which may be the same as or different from the polynucleotide ofinterest in the producer vector). In such cases it may be desirable touse a carrier vector that is defective for systemic infection, e.g.,because it lacks one or more necessary cis-acting sequences, in order tominimize spread of the recombinant carrier vector to non-target plants.

The carrier vector may (but need not) include a cell-to-cell movementcomponent (e.g., a gene encoding a cell-to-cell movement protein or anoncoding component that is needed for cell-to-cell movement) and/or maylack one or more replicase protein encoding components. In embodimentsin which the carrier vector does not include a cell-to-cell movementcomponent (e.g., a functional MP encoding portion), such a componentshould be included in the producer vector.

A complete vector set includes all components necessary for successfulsystemic viral infection and expression of a polynucleotide of interest.The term “component” is intended to include both protein codingsequences and non-coding sequences such as cis-acting sequences (e.g.,promoters, origin of assembly, portions corresponding to untranslatedregions in mRNA). Different vectors, or vector elements, may be derivedfrom different plant viruses. In fact, it may be desirable to preparevectors from elements of different viruses in order to take advantage ofdifferent viral characteristics (e.g., host range, promoter activitylevel, virion dimensions, etc.).

In some embodiments, a producer vector is provided that includes apolynucleotide of interest, a replicase gene, and a movement proteingene, but lacks a functional coat protein encoding component, and acarrier vector is provided that expresses a coat protein gene. Forexample, a producer vector may include a TMV-based vector in which theTMV CP coding sequence has been replaced by a polynucleotide ofinterest, under control of the TMV CP promoter. This producer vector isunable to move systemically. A wild type AlMV vector can serve as thecarrier vector. The AlMV vector contains a functional coat proteinencoding component. Co-infection with both producer and carrier vectorsallows the CP produced from the AlMV vector CP coding sequence tocomplement the TMV-based vector, resulting in systemic movement of theTMV-based vector and expression of the polynucleotide in leaves thatwere not initially infected. Alternately, an AlMV-based vector in whichone or more viral components other than those required for expression ofAlMV CP has been removed can be used (e.g., an AlMV-based vector lackingfunctional MP or replication protein coding components), provided thatfunctional CP coding sequences and an operably linked promoter arepresent. The CP can be from AlMV or from another virus.

In some embodiments, the CP allows for systemic movement of the carriervector, while in other embodiments a CP is selected that does not allowfor systemic movement of the carrier vector but does allow for systemicmovement of the producer vector. In those embodiments in which thecarrier vector lacks one or more of the viral components other thanthose required for expression of AlMV CP, the producer vector maycomplement the carrier vector. For example, the producer vector maysupply a component such as a functional MP or replicase protein codingsequence that allows for cell-to-cell movement or replication,respectively, of the carrier vector (and, in some cases, also theproducer vector). It will be appreciated that where either the produceror the carrier is lacking a replication protein encoding component(e.g., a functional RNA polymerase coding component) and the othervector (carrier or producer, respectively) supplies the missingcomponent, it will often be desirable to insert a promoter (e.g., agenomic promoter) from the vector that supplies the functionalreplication component into the vector lacking the functional replicationprotein coding component in order to achieve effectivetrans-complementation of replication function.

Another example of a useful viral vector system includes a producervector in which a polynucleotide of interest is inserted into an AlMVvector, replacing the native AlMV CP encoding component. Thepolynucleotide of interest is placed under control of the AlMV CPpromoter. This producer vector is incapable of systemic infection sinceit lacks CP but is able to replicate and move cell-to-cell within aninfected leaf. The system also includes a cauliflower mosaic virus(CMV)-based carrier vector in which an AlMV CP encoding portion, with orwithout the AlMV CP 3′ UTR is inserted into a CMV vector, replacing theCMV CP encoding component found in the genome of naturally occurringCMV. The AlMV CP encoding component is placed under control of the CMVCP promoter. This vector expresses AlMV CP. Co-infection with theproducer and carrier vectors allows CP expressed from the carrier vectorto trans-complement the producer vector's lack of functional CP encodingcomponents, allowing systemic movement of the producer vector. The AlMVCP also allows systemic movement of the carrier vector.

In some embodiments, it can be desirable to insert a portion of codingor noncoding sequence from the carrier vector into the producer vector,or vice versa. For example, certain sequences may enhance replication orfacilitate cell-to-cell or long distance movement. In particular,certain sequences may serve as recognition sites for formation of acomplex between a viral transcript and a CP (e.g., an origin ofassembly). In such a case, if systemic movement of a first viral vectoris to be achieved using CP provided in trans from a second viral vector,it may be desirable to insert such sequences from the second viralvector that facilitate activity of the CP into the first viral vector.Such sequences may include, for example, part or all of a viraltranscript 3′ UTR. In some cases, part or all of the RNA3 3′ UTR of AlMVis inserted into a different viral vector, e.g., a TMV-based vector.Including this component in the TMV-based vector facilitates the abilityto AlMV CP to trans-complement a TMV-based vector that lacks afunctional TMV CP encoding portion. It will be appreciated that thisgeneral principle may be applied to any viral vector system comprisingtrans-complementing vectors, e.g., trans-complementing producer andcarrier vector systems.

As will be appreciated by those of ordinary skill in the art, so long asa vector set includes a producer vector that is incapable of systemicviral infection (e.g., lacking one or more functional replicationprotein, movement protein, or coat protein encoding components) and acarrier vector that provides the function(s) lacking in the producervector, that set is appropriate for use in accordance with the methodsdescribed herein. In some embodiments, no individual vector is capableof systemic viral infection but, as a set, one or both of the vectorsis/are competent for such infection and expression of the polynucleotideof interest. Such a system can offer a number of advantages. Forexample, it will be appreciated that if the producer vector infects aplant in the absence of the carrier vector, no systemic infection willresult. This diminishes the risk that the polynucleotide of interestwill be expressed in unintended (non-target) plants, even of the samespecies as the target plant. In particular, if the carrier vector is notcompetent for replication or cell-to-cell movement (because it lacks acomponent required for replication or cell-to-cell movement) or if it isincompetent for systemic infection (e.g., because it lacks a cis-actingsequence such as an origin of assembly that is required for longdistance movement), the likelihood that both producer and carriervectors will co-infect an unintended plant host are greatly reduced.

Generally, in order to preserve viral function and also simply for easeof genetic manipulation, vectors are prepared by altering an existingplant virus genome (e.g., by removing particular genes and/or bydisrupting or substituting particular sequences so as to inactivate orreplace them). In such circumstances, the vectors will show very highsequence identity with natural viral genomes. Of course, completelynovel vectors may also be prepared, for example, by separately isolatingindividual desired genetic elements and linking them together,optionally with the inclusion of additional elements. Also, it should benoted that where a particular vector is said to lack a given gene,protein, or activity (e.g., the producer vector lacks a coat proteingene), it is sufficient if no such protein or activity is expressed fromthe vector under conditions of infection, even though the vector maystill carry the relevant coding sequence. In general, however, it istypically desirable to remove the relevant coding sequences from thevector.

Analogously, when a vector is said to affirmatively express a particularprotein or activity, it is not necessary that the relevant gene beidentical to the corresponding gene found in nature. For instance, ithas been found that the coat protein can sometimes tolerate smalldeletions (see, for example, WO 00/46350, which is incorporated hereinby reference in its entirety). So long as the protein is functional, itmay be used in accordance with the methods described herein. Very highsequence identity with the natural protein, however, is generallyconsidered to be most useful. For instance, large deletions (e.g.,greater than about 25 amino acids) generally should be avoided.Typically, viral proteins will show at least 50% (e.g., 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequenceidentity with the corresponding natural viral protein. Moreparticularly, the viral protein typically should have 100% identity withcritical functional portions (typically of at least several amino acids,often of at least 10, 20, 30, 40, 50 or more amino acids) of therelevant natural viral protein.

It is noted that in the case of many proteins, a number of amino acidchanges can be made without significantly affecting the functionalactivity and/or various other properties of the protein such asstability, etc. In particular, many proteins tolerate conservative aminoacid changes—the substitution of an amino acid with a different aminoacid having similar properties, without significant reduction inactivity. Conservative amino acid substitution is well known in the artand represents one approach to obtaining a polypeptide having similar orsubstantially similar properties to those of a given polypeptide whilealtering the amino acid sequence. In general, amino acids have beenclassified and divided into groups according to (1) charge (positive,negative, or uncharged); (2) volume and polarity; (3) Grantham'sphysico-chemical distance; and combinations of these. See, e.g., Zhang,J. Mol. Evol., 50:56-68, 2000; Grantham, Science, 85:862-864, 1974;Dagan et al., Mol. Biol. Evol., 19(7), 1022-1025, 2002; Biochemistry,4th Ed., Stryer et al., W. Freeman and Co., 1995; and U.S. Pat. No.6,015,692. For example, amino acids may be divided into the followingcategories based on volume and polarity: special (C); neutral and small(A, G, P, S, T); polar and relatively small (N, D, Q, E), polar andrelatively large (R, H, K), nonpolar and relatively small (I, L, M, V),and nonpolar and relatively large (F, W, Y). A conservative amino acidsubstitution may be defined as one that replaces one amino acid with anamino acid in the same group. Thus a variety of functionally equivalentproteins can be derived by making one or more conservative amino acidsubstitutions in a given viral protein.

Any plant susceptible to viral infection may be utilized in accordancewith the methods described herein. In general, it will often bedesirable to utilize plants that are amenable to growth under definedconditions, for example in a greenhouse and/or in aqueous systems. Italso may be desirable to select plants that are not typically consumedby human beings or domesticated animals and/or are not typically part ofthe human food chain, so that they may be grown outside without concernthat the expressed polynucleotide may be undesirably ingested. In otherembodiments, however, it will be desirable to employ edible plants.

Often, certain desirable plant characteristics will be determined by theparticular polynucleotide to be expressed. To give but a few examples,when the polynucleotide encodes a protein to be produced in high yield(as will often be the case, for example, when therapeutic proteins areto be expressed), it will often be desirable to select plants withrelatively high biomass (e.g., tobacco, which has the additionaladvantages that it is highly susceptible to viral infection, has a shortgrowth period, and is not in the human food chain). Where thepolynucleotide encodes a protein whose full activity requires (or isinhibited by) a particular post-translational modification, the ability(or inability) of certain plant species to accomplish the relevantmodification (e.g., a particular glycosylation) may direct selection.

In some embodiments, crop plants, or crop-related plants, are utilized.In some embodiments, edible plants are utilized.

Plants suitable for use in accordance with the methods described hereininclude, without limitation, Angiosperms, Bryophytes (e.g., Hepaticae,Musci, etc.), Pteridophytes (e.g., ferns, horsetails, lycopods),Gymnosperms (e.g., conifers, cycase, Ginko, Gnetales), and Algae (e.g.,Chlorophyceae, Phaeophyceae, Rhodophyceae, Myxophyceae, Xanthophyceae,and Euglenophyceae). In some embodiments, members of the familyLeguminosae (Fabaceae; e.g., pea, alfalfa, soybean); Gramineae (Poaceae;e.g., corn, wheat, rice); Solanaceae, particularly of the genusLycopersicon (e.g., tomato), Solanum (e.g., potato, eggplant), Capsium(e.g., pepper), or Nicotiana (e.g., tobacco); Umbelliferae, particularlyof the genus Daucus (e.g., carrot), Apium (e.g., celery), or Rutaceae(e.g., oranges); Compositae, particularly of the genus Lactuca (e.g.,lettuce); and Brassicaceae (Cruciferae), particularly of the genusBrassica or Sinapis, can be particularly useful. For example, usefulBrassicaceae family members include Brassica campestris, B. carinata, B.juncea, B. napus, B. nigra, B. oleraceae, B. tournifortii, Sinapis alba,and Raphanus sativus.

The expression system may be employed to infect, and/or to express apolynucleotide in plants at any stage of development including, forexample, mature plants, seedlings, sprouts, and seeds. The system may beemployed to infect any part of a plant (e.g., roots, leaves, stems,etc.). In some embodiments, the system is used to infect sprouts.Generally, a plant is considered to be a sprout when it is a seedlingthat does not require external nutrients or energy in the form of lightor heat beyond what is required to achieve normal germinationtemperatures. Often, a seedling that is less than two weeks old, andtypically less than 10 days old, is considered to be a sprout.

In general, viral vectors may be delivered to plants according to knowntechniques. For example, the vectors themselves may be directly appliedto plants (e.g., via abrasive inoculations, mechanized sprayinoculations, vacuum infiltration, particle bombardment, orelectroporation). Alternatively, virions may be prepared (e.g., fromalready infected plants), and may be applied to other plants accordingto known techniques.

As noted above, in some embodiments, viral vectors are applied tosprouts (e.g., through infiltration or mechanical inoculation [spray]).

Where infection is to be accomplished by direct application of a viralgenome to a plant, any available technique may be used to prepare thegenome. For example, many viruses that are usefully employed inaccordance with the methods described herein have ssRNA genomes. ssRNAmay be prepared by transcription of a DNA copy of the genome, or byreplication of an RNA copy, either in vivo or in vitro. Given thereadily availability of easy-to-use in vitro transcription systems(e.g., SP6, T7, reticulocyte lysate, etc.), and also the convenience ofmaintaining a DNA copy of an RNA vector, it is expected that ssRNAvectors will often be prepared by in vitro transcription, particularlywith T7 or SP6 polymerase.

In some embodiments, it will be desirable to isolate polynucleotideexpression products from the plant tissues that express them. It alsomay be desirable to formulate such isolated products for their intendeduse (e.g., as a pharmaceutical or diagnostic agents, as reagents, etc.).In other embodiments, it will be desirable to formulate the productstogether with some or all of the plant tissues that express them.

To isolate the expression product from some or all of the plant tissuethat expresses it, any available purification techniques may beemployed. Those of ordinary skill in the art are familiar with a widerange of fractionation and separation procedures (see, for example,Scopes et al., Protein Purification: Principles and Practice, 3^(rd)Ed., Janson et al., “Protein Purification: Principles, High ResolutionMethods, and Applications,” Wiley-VCH, 1998; Springer-Verlag, NY, 1993;and Roe, Protein Purification Techniques, Oxford University Press, 2001,each of which is incorporated herein by reference in its entirety).Often, it will be desirable to render the product more than about 50%,preferably more than about 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% pure.

To formulate the product together with the plant material, it will oftenbe desirable to have utilized a plant that is not toxic to the relevantrecipient (e.g., a human or other animal). Relevant plant tissue (e.g.,leaves) may simply be harvested and processed according to techniquesknown in the art, with due consideration to maintaining activity of theexpressed product. In some embodiments, the polynucleotide can beexpressed in an edible plant (and, specifically in edible portions ofthe plant) so that the material can subsequently be eaten. For example,where the polynucleotide encodes a nutritionally relevant protein or atherapeutic protein that is active after oral delivery (when properlyformulated), it may be useful to produce the protein in an edible plantportion, and to formulate the expressed polynucleotide for oral deliverytogether with some or all of the plant material with which thepolynucleotide was expressed.

Where the polynucleotide encodes or produces a therapeutic agent, it maybe formulated according to known techniques. For example, an effectiveamount of a pharmaceutically active product can be formulated togetherwith one or more organic or inorganic, liquid or solid, pharmaceuticallysuitable carrier materials. A pharmaceutically active product may beemployed in dosage forms such as tablets, capsules, troches,dispersions, suspensions, solutions, capsules, creams, ointments,aerosols, powder packets, liquid solutions, solvents, diluents, surfaceactive agents, isotonic agents, thickening or emulsifying agents,preservatives, and solid bindings, as long as the biological activity ofthe protein is not destroyed by such dosage form.

Materials that can serve as pharmaceutically acceptable carriersinclude, but are not limited to, sugars such as lactose, glucose andsucrose; starches such as corn starch and potato starch; cellulose andits derivatives such as sodium carboxymethyl cellulose, ethyl celluloseand cellulose acetate; powdered tragacanth; malt; gelatin; talc;excipients such as cocoa butter and suppository waxes; oils such aspeanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, cornoil and soybean oil; glycols such a propylene glycol; esters such asethyl oleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening agents, flavoringagents, and perfuming agents, preservatives, and antioxidants can alsobe present in the composition, according to the judgment of theformulator (see also Remington's Pharmaceutical Sciences, FifteenthEdition, E. W. Martin, Mack Publishing Co., Easton Pa., 1975). Forexample, a polynucleotide expression product may be provided as apharmaceutical composition by means of conventional mixing granulatingdragee-making, dissolving, lyophilizing, or similar processes.

In some embodiments, it may be useful to prolong the effect of apharmaceutical preparation by slowing the absorption of thepharmaceutically active product (e.g., protein) that is subcutaneouslyor intramuscularly injected. This may be accomplished by the use of aliquid suspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the product then depends upon itsrate of dissolution, which in turn, may depend upon size and form.Alternatively, delayed absorption of a parenterally administered productis accomplished by dissolving or suspending the product in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the protein in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of product topolymer and the nature of the particular polymer employed, the rate ofrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations may be prepared by entrapping the product in liposomes ormicroemulsions, which are compatible with body tissues.

Enterically administered preparations of pharmaceutically activeproducts may be introduced in solid, semi-solid, suspension or emulsionform and may be compounded with any pharmaceutically acceptablecarriers, such as water, suspending agents, and emulsifying agents. Theexpression products may also be administered by means of pumps orsustained-release forms, especially when administered as a preventivemeasure, so as to prevent the development of disease in a subject or toameliorate or delay an already established disease.

Pharmaceutically active products, optionally together with plant tissue,can be particularly well suited for oral administration aspharmaceutical compositions. Harvested plant material may be processedin any of a variety of ways (e.g., air drying, freeze drying, extractionetc.), depending on the properties of the desired therapeutic productand its desired form. In preferred embodiments, such compositions asdescribed above are ingested orally alone or ingested together with foodor feed or a beverage. Compositions for oral administration includeinfected plants; extractions of the infected plants, and proteinspurified from infected plants provided as dry powders, foodstuffs,aqueous or non-aqueous solvents, suspensions, or emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oil, fish oil, and injectable organic esters. Aqueous carriersinclude water, water-alcohol solutions, emulsions or suspensions,including saline and buffered medial parenteral vehicles includingsodium chloride solution, Ringer's dextrose solution, dextrose plussodium chloride solution, Ringer's solution containing lactose or fixedoils. Examples of dry powders include any infected plant biomass thathas been dried, for example, freeze dried, air dried, or spray dried.For example, the plants may be air dried by placing them in a commercialair dryer at about 120 degrees Fahrenheit until the biomass containsless than 5% moisture by weight. The dried plants may be stored forfurther processing as bulk solids or further processed by grinding to adesired mesh sized powder. Alternatively, freeze-drying may be used forproducts that are sensitive to air-drying. Products may be freeze driedby placing them into a vacuum drier and dried frozen under a vacuumuntil the biomass contains less than about 5% moisture by weight. Thedried material can be further processed.

Infected plants may be administered as or together with one or moreherbal preparations. Useful herbal preparations include liquid and solidherbal preparations. Some examples of herbal preparations includetinctures, extracts (e.g., aqueous extracts, alcohol extracts),decoctions, dried preparations (e.g., air-dried, spray dried, frozen, orfreeze-dried), powders (e.g., lyophilized powder), and liquid. Herbalpreparations can be provided in any standard delivery vehicle, such as acapsule, tablet, suppository, liquid dosage, etc. Those skilled in theart will appreciate the various formulations and modalities of deliveryof herbal preparations that may be applied.

Those skilled in the art will also appreciate that a particularly usefulmethod of obtaining the desired pharmaceutically active products is byextraction. Infected plants may be extracted to remove the desiredproducts from the residual biomass, thereby increasing the concentrationand purity of the product. Plants also may be extracted in a bufferedsolution. For example, the fresh harvested plants may be transferredinto an amount of ice-cold water at a ratio of one to one by weight thathas been buffered with, e.g., phosphate buffer. Protease inhibitors canalso be added as required. The plants can be disrupted by vigorousblending or grinding while suspended in the buffer solution and theextracted biomass removed by filtration or centrifugation. The transgeneproduct carried in solution can be further purified by additional stepsor converted to a dry powder by freeze-drying or precipitation.Extraction can also be carried out by pressing. Live plants can also beextracted by pressing in a press or by being crushed as they are passedthrough closely spaced rollers. The fluids expressed from the crushedplants are collected and processed according to methods well known inthe art. Extraction by pressing allows the release of the products in amore concentrated form. However, the overall yield of the product may belower than if the product were extracted in solution.

Infected plants, extractions, powders, dried preparations and purifiedprotein products, etc., can also be in encapsulated form with or withoutone or more excipients as noted above. The solid dosage forms oftablets, dragees, capsules, pills, and granules can be prepared withcoatings and shells such as enteric coatings, release controllingcoatings and other coatings well known in the pharmaceutical formulatingart. In such solid dosage forms the active product may be admixed withat least one inert diluent such as sucrose, lactose or starch. Suchdosage forms may also comprise, as is normal practice, additionalsubstances other than inert diluents, e.g., tableting lubricants andother tableting aids such a magnesium stearate and microcrystallinecellulose. In the case of capsules, tablets and pills, the dosage formsmay also comprise buffering agents. They may optionally containopacifying agents and can also be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain part of theintestinal tract, optionally, in a delayed manner. Examples of embeddingcompositions that can be used include polymeric substances and waxes.

In some embodiments, an infected plant expressing a pharmaceuticallyactive product, or biomass of an infected plant, is administered orallyas medicinal food. Such edible compositions are consumed by eating raw,if in a solid form, or by drinking, if in liquid form. In someembodiments, the transgenic plant material is directly ingested withouta prior processing step or after minimal culinary preparation. Forexample, the pharmaceutically active protein is expressed in a sproutthat can be eaten directly (e.g., an alfalfa sprout, mung bean sprout,or a spinach or lettuce leaf sprout). In some embodiments, the plantbiomass is processed and the material recovered after the processingstep is ingested.

Therapeutic Methods and Pharmaceutical Compositions

The C-terminal endostatin polypeptides disclosed herein, or nucleicacids encoding the C-terminal endostatin polypeptides, can be used totreat fibrosis. In several examples, the C-terminal endostatinpolypeptides, or nucleic acid encoding these polypeptides are of use todecrease fibrosis, such as in a subject. Thus, in several embodiments,the methods include administering to a subject a therapeuticallyeffective amount of one or more of the C-terminal endostatinpolypeptides disclosed herein, or polynucleotides encoding thesepolypeptides, in order to decrease fibrosis. In some examples, theC-terminal endostatin polypeptide comprises or consists of amino acids133-180 of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:13, or SEQ ID NO:14.However, any of the C-terminal endostatin polypeptides disclosed hereincan be used to decrease fibrosis. In some embodiments, the peptides canbe administered as a unit dose. In some embodiments, the polypeptidesare administered as multimers.

Suitable subjects include those with a fibrosis of the skin or lungs,but fibrosis of any tissue can be treated using the methods disclosedherein. In one example, the subject has scleroderma. In other examples,the subject has idiopathic pulmonary fibrosis, morphea, fibrosis as aresult of Graft-Versus-Host Disease (GVHD), a keloid or hypertrophicscar, subepithelial fibrosis, endomyocardial fibrosis, uterine fibrosis,myelofibrosis, retroperitoneal fibrosis, nephrogenic systemic fibrosis,scarring after surgery, asthma, cirrhosis/liver fibrosis, aberrant woundhealing, glomerulonephritis, and multifocal fibrosclerosis.

In further examples, the methods are used to treat the systemic form ofscleroderma, such as limited cutaneous scleroderma or diffuse cutaneousscleroderma (or systemic sclerosis). The methods can be used to treatthe localized form of scleroderma, including morphea and linearscleroderma.

The methods can include selecting a subject in need of treatment, suchas a subject with a fibrotic disease, such as scleroderma, idiopathicpulmonary fibrosis, morphea, a keloid scar, a hypertrophic scar, orsubepithelial fibrosis. In exemplary applications, compositions areadministered to a subject having a fibrotic disease, such asscleroderma, idiopathic pulmonary fibrosis, morphea, a keloid scar, ahypertrophic scar, or subepithelial fibrosis, or any of the disorderslisted above, in an amount sufficient to reduce the fibrosis. Amountseffective for this use will depend upon the severity of the disease, thegeneral state of the patient's health, and the robustness of thepatient's immune system. In one example, a therapeutically effectiveamount of the compound is that which provides either subjective reliefof a symptom(s) or an objectively identifiable improvement as noted bythe clinician or other qualified observer.

A method is provided herein for decreasing skin thickness. The methodincludes administering a therapeutically effective amount of aC-terminal endostatin polypeptide, thereby decreasing skin thickness. Inanother embodiment, a method is provided for decreasing lung fibrosis.The method includes administering a therapeutically effective amount ofa C-terminal endostatin polypeptide, thereby decreasing skin thickness.Any of the C-terminal endostatin polypeptides disclosed herein can beused in these methods. In some embodiments, the C-terminal endostatinpolypeptide comprises, or consists of, amino acids 133-180 of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:13, or SEQ ID NO:14.

Methods are provided herein for decreasing lysyl oxidase (LOX), such astransforming growth factor (TGF)-β induced LOX. The method includescontacting a cell with an effective amount of a C-terminal endostatinpolypeptide, thereby decreasing LOX. The methods can be practiced invivo or in vitro. In some embodiments, the methods include comparing theamount of LOX produced by a cell contacted with a C-terminal endostatinpolypeptide to a control. The control can be a standard value, or theamount of LOX produced by a cell not contacted with the C-terminalendostatin polypeptide, such as a cell contacted with a carrier.

Methods are provided herein for increasing matrix metalloproteinase-2(MMP-2). The method includes contacting a cell with an effective amountof a C-terminal endostatin polypeptide, thereby increasing MMP-2production. The methods can be practiced in vivo or in vitro. In someembodiments, the methods include comparing the amount of MMP-2 producedby a cell contacted with a C-terminal endostatin polypeptide to acontrol The control can be a standard value, or the amount of MMP-2produced by a cell not contacted with the C-terminal endostatinpolypeptide, such as a cell contacted with a carrier.

A C-terminal endostatin polypeptide can be administered by any meansknown to one of skill in the art (see Banga, “Parenteral ControlledDelivery of Therapeutic Peptides and Proteins,” in Therapeutic Peptidesand Proteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995)either locally or systemically, such as by intradermal, intrathecal,intramuscular, subcutaneous, intraperitoneal or intravenous injection,but even oral, nasal, transdermal or anal administration iscontemplated. In some embodiments, administration is by subcutaneous,intradermal, or intramuscular injection. In another embodiment,administration is by intraperitoneal or intrathecal administration. Toextend the time during which the peptide or protein is available tostimulate a response, the peptide or protein can be provided as animplant, an oily injection, or as a particulate system. The particulatesystem can be a microparticle, a microcapsule, a microsphere, ananoparticle, a nanocapsule, or similar particle. (see, e.g., Banga,supra).

For treatment of the skin, a therapeutically effective amount of atleast one C-terminal endostatin polypeptide, or a nucleic acid encodingthe peptide, can be locally administered to the affected area of theskin, such as in the form of an ointment. In some embodiments, theointment is an entirely homogenous semi-solid external agent with afirmness appropriate for easy application to the skin. Such an ointmentcan include fats, fatty oils, lanoline, VASELINE®, paraffin, wax, hardointments, resins, plastics, glycols, higher alcohols, glycerol, wateror emulsifier and a suspending agent. Using these ingredients as a base,a decoy compound can be evenly mixed. Depending on the base, the mixturecan be in the form of an oleaginous ointment, an emulsified ointment, ora water-soluble ointment oleaginous ointments use bases such as plantand animal oils and fats, wax, VASELINE® and liquid paraffin. Emulsifiedointments are comprised of an oleaginous substance and water, emulsifiedwith an emulsifier. They can take either an oil-in-water form (O/W) or awater-in-oil-form (W/O). The oil-in-water form (O/W) can be ahydrophilic ointment. The water-in-oil form (W/O) initially lacks anaqueous phase and can include hydrophilic VASELINE® and purifiedlanoline, or it can contain a water-absorption ointment (including anaqueous phase) and hydrated lanoline. A water-soluble ointment cancontain a completely water-soluble Macrogol base as its main ingredient.

Pharmaceutically acceptable carriers include a petroleum jelly, such asVASELINE®, wherein the petroleum jelly contains 5% stearyl alcohol, orpetroleum jelly alone, or petroleum jelly containing liquid paraffin.Such carriers enable pharmaceutical compositions to be prescribed informs appropriate for consumption, such as tablets, pills, sugar-coatedagents, capsules, liquid preparations, gels, ointments, syrups,slurries, and suspensions. When locally administered into cells in anaffected area or a tissue of interest, the at least one C-terminalendostatin polypeptide, or polynucleotide encoding the peptide can beadministered in a composition that contains a synthetic or naturalhydrophilic polymer as the carrier. Examples of such polymers includehydroxypropyl cellulose and polyethylene glycol. One or more C-terminalendostatin polypeptides, or polynucleotide encoding the polypeptides,can be mixed with a hydrophilic polymer in an appropriate solvent. Thesolvent is then removed by methods such as air-drying, and the remainderis then shaped into a desired form (for example, a sheet) and applied tothe target site. Formulations containing such hydrophilic polymers keepwell as they have a low water-content. At the time of use, they absorbwater, becoming gels that also store well. In the case of sheets, thefirmness can be adjusted by mixing a polyhydric alcohol with ahydrophilic polymer similar to those above, such as cellulose, starchand its derivatives, or synthetic polymeric compounds. Hydrophilicsheets thus formed can be used. A therapeutically effective amount ofone or more C-terminal endostatin polypeptide, or polynucleotideencoding the peptide can also be incorporated into bandages anddressings.

For administration by inhalation, the C-terminal endostatin polypeptide,or polynucleotide encoding the peptide can be conveniently delivered inthe form of an aerosol spray presentation from pressurized packs or anebulizer, with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesfor use in an inhaler or insufflator can be formulated containing apowder mix of the compound and a suitable powder base such as lactose orstarch.

In some embodiments, the C-terminal endostatin polypeptide, such as, butnot limited to E4, can be administered by inhalation. For example, theC-terminal endostatin polypeptide can be administered in an aerosolizedform, such as using a nebulizer or a metered dose inhaler. Technologiesof use include micropump nebulizers (such as the AEROGEN GO® system),jet nebulizers designed to produce large fine particle fractions (suchas the PARI LC STAR®), jet nebulizers developing less shear duringatomization (such as the HUDSON MICROMIST®), and ultrasonic nebulizers(such as the DeVilbiss ULTRA-NEB®).

The endostatin polypeptide can be dissolved in a carrier, such assaline, and atomized using the devices above. The associated aerosolscan be collected using a NEXT GENERATION IMPACTOR® (NGI) (MSP Corp.,Shoreview, Minn.), which uses a series of aerodynamic stages to separateand collect the aerosol into separate fractions based on droplet size.Since droplet size is the primary determinant of deposition location inthe lungs, this device allows us to specifically isolate the portion ofthe liquid aerosol that will deposit in the small airways and alveoli.

Aerosol particle size is often expressed in terms of mass medianaerodynamic diameter (MMAD), a parameter that is based on particle size,shape, and density. For a spherical particle, MMAD is equal to MMD(p^(1/2)), in which MMD is mass median diameter and r is the bulkdensity. For a non-spherical particle, MMAD is equal to MMD (p/x)^(1/2),in which X is the shape factor. Thus, particles with larger than unitdensity will have actual diameters smaller than their MMAD.

The site of particle deposition within the respiratory tract isdemarcated based on particle size. In one example, particles of about 1to about 500 microns are utilized, such as particles of about 25 toabout 250 microns, or about 10 to about 25 microns are utilized. Inother embodiments, particles of about 1 to 50 microns are utilized. Foruse in a metered dose inhaler, for administration to lungs particles ofless than about 10 microns, such as particles of about 2 to about 8microns, such as about 1 to about 5 microns, such as particles of 2 to 3microns, can be utilized.

A therapeutically effect amount of a C-terminal endostatin polypeptide,or polynucleotide encoding the peptide can be administered in thepharmaceutically acceptable carrier. Pharmacologically acceptablecarriers (e.g., physiologically or pharmaceutically acceptable carriers)are well known in the art, and include, but are not limited to bufferedsolutions as a physiological pH (e.g. from a pH of about 7.0 to about8.0, or at a pH of about 7.4). One specific, non-limiting example of aphysiologically compatible buffered solution is phosphate bufferedsaline. Other pharmacologically acceptable carriers include penetrants,which are particularly suitable for pharmaceutical formulations that areintended to be topically applied (for example in the application ofsurgical wounds to promote healing).

The pharmacological compositions disclosed herein facilitate the use ofat least one C-terminal endostatin polypeptide, or polynucleotideencoding the peptide, either in vivo or ex vivo, to decrease fibrosis.Such a composition can be suitable for delivery of the active ingredientto any suitable subject, and can be manufactured in a manner that isitself known, e.g., by means of conventional mixing, dissolving,granulating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Pharmacological compositions can be formulated in aconventional manner using one or more pharmacologically (e.g.,physiologically or pharmaceutically) acceptable carriers, as well asoptional auxiliaries that facilitate processing of the active compoundsinto preparations which can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen. Thus, forinjection, the active ingredient can be formulated in aqueous solutions.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, a therapeutically effective amount of at leastone C-terminal endostatin polypeptide, or a nucleic acid encoding thepeptide, can be combined with carriers suitable for incorporation intotablets, pills, capsules, liquids, gels, syrups, slurries, suspensionsand the like.

Protein drugs are subject to protease-mediated degradation in thedigestive tract through the action of enzymes such as trypsin,chymotrypsin, and brush border peptidases, such that oral administrationof large protein molecules often does not result in the intendedtherapeutic effect (Soltero and Ekwruibe, 2001 Innovations inPharmaceutical Technology, 1:106-110). Surprisingly, the C-terminalendostatin polypeptide described herein can maintain its activity andtherapeutic effects even when administered via an oral route. Withoutbeing bound by theory, it is believed that the C-terminal endostatinpolypeptide described herein includes additional properties that allowit to escape protease activity and become adsorbed in the smallintestine and enter circulation to provide therapeutic benefit.

In some embodiments, for parenteral administration, a therapeuticallyeffective amount of at least one C-terminal endostatin polypeptide, or anucleic acid encoding the peptide, can be administered by injection,such as by bolus injection or continuous infusion. Such compositions cantake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Other pharmacological excipientsare known in the art.

Optionally, the at least one C-terminal endostatin polypeptide, orpolynucleotide encoding the peptide can be contained within orconjugated with a heterologous protein, hydrocarbon or lipid, whetherfor in vitro or in vivo administration. Co-administration can be suchthat the at least one C-terminal endostatin polypeptide, orpolynucleotide encoding the peptide is administered before, atsubstantially the same time as, or after the protein, hydrocarbon, orlipid. In some embodiments, the at least one C-terminal endostatinpolypeptide, or polynucleotide encoding the peptide is administered atsubstantially the same time, as the protein, hydrocarbon, or lipid.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compositions of the invention described above,increasing convenience to the subject and the physician. Many types ofrelease delivery systems are available and known to those of ordinaryskill in the art. They include polymer based systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems, such as lipids including sterols suchas cholesterol, cholesterol esters and fatty acids or neutral fats suchas mono- di- and tri-glycerides; hydrogel release systems; silasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which the at least one C-terminal endostatin polypeptide, orpolynucleotide encoding the peptide is contained in a form within amatrix such as those described in U.S. Pat. Nos. 4,452,775; 4,667,014;4,748,034; 5,239,660; and 6,218,371 and (b) diffusional systems in whichan active component permeates at a controlled rate from a polymer suchas described in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition,pump-based hardware delivery systems can be used, some of which areadapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions, such as scleroderma.Long-term release, as used herein, means that the implant is constructedand arranged to deliver therapeutic levels of the active ingredient forat least 30 days, and preferably 60 days. Long-term sustained releaseimplants are well known to those of ordinary skill in the art andinclude some of the release systems described above. These systems havebeen described for use with oligodeoxynucleotides (see U.S. Pat. No.6,218,371). For use in vivo, nucleic acids and peptides are preferablyrelatively resistant to degradation (such as via endo- andexo-nucleases). Thus, modifications, such as the inclusion of aC-terminal amide, can be used.

The therapeutically effective amount of C-terminal endostatinpolypeptide, or polynucleotide encoding the peptide will be dependent onthe C-terminal endostatin polypeptide, or polynucleotide encoding thepeptide that is utilized, the subject being treated, the severity andtype of the affliction, and the manner of administration. For example, atherapeutically effective amount of a polynucleotide encoding thepeptide can vary from about 0.01 μg per kilogram (kg) body weight toabout 1 g per kg body weight, such as about 1 μg to about 5 mg per kgbody weight, or about 5 μg to about 1 mg per kg body weight. The exactdose is readily determined by one of skill in the art based on thepotency of the specific compound the age, weight, sex and physiologicalcondition of the subject.

With regard to the administration of nucleic acids, one approach toadministration of nucleic acids is direct treatment with plasmid DNA,such as with a mammalian expression plasmid. As described above, thenucleotide sequence encoding a N-terminal endostatin peptide can beplaced under the control of a promoter to increase expression of themolecule.

When a viral vector is utilized for administration in vivo, it isdesirable to provide the recipient with a dosage of each recombinantvirus in the composition in the range of from about 10⁵ to about 10¹⁰plaque forming units/mg mammal, although a lower or higher dose can beadministered. The composition of recombinant viral vectors can beintroduced into a mammal either prior to any evidence of a cancer, or tomediate regression of the disease in a mammal afflicted with the cancer.Examples of methods for administering the composition into mammalsinclude, but are not limited to, exposure of cells to the recombinantvirus ex vivo, or injection of the composition into the affected tissueor intravenous, subcutaneous, intradermal or intramuscularadministration of the virus. Alternatively the recombinant viral vectoror combination of recombinant viral vectors may be administered locallyby direct injection into the cancerous lesion in a pharmaceuticallyacceptable carrier. Generally, the quantity of recombinant viral vector,carrying the nucleic acid sequence of one or more C-terminal endostatinpolypeptides to be administered is based on the titer of virusparticles. An exemplary range of the immunogen to be administered is 10⁵to 10¹⁰ virus particles per mammal, such as a human.

In one specific, non-limiting example, a pharmaceutical composition forintravenous administration would include about 0.1 μg to 10 mg ofC-terminal endostatin polypeptide per patient per day. Dosages from 0.1up to about 100 mg per patient per day can be used, particularly if theagent is administered to a secluded site and not into the circulatory orlymph system, such as into a body cavity or into a lumen of an organ.Actual methods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remington's Pharmaceutical Sciences, 19^(th) Ed.,Mack Publishing Company, Easton, Pa., 1995.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thesubject. In some embodiments, the dosage is administered once as abolus, but in another embodiment can be applied periodically until atherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of disease without producingunacceptable toxicity to the subject. Systemic or local administrationcan be utilized.

In a further method, an additional agent is administered. In oneexample, this administration is sequential. In other examples, theadditional agent is administered simultaneously with the C-terminalendostatin polypeptide.

For the treatment of scleroderma, examples of additional agents that canbe used with a C-terminal endostatin polypeptides include nifedipine,amlodipine, diltiazem, felodipine, or nicardipine. An investigationaldrug GLEEVEC®, is also used for the treatment of scleroderma. GLEEVEC®or other tyrosine kinase inhibitors can be used with the C-terminalendostatin polypeptides disclosed herein. Patients with lung involvementof scleroderma benefit from oxygen therapy; the C-terminal endostatinpolypeptides disclosed herein can be administered with this therapy.

For the treatment of fibrosis of the skin and scleroderma, additionalagents of use are d-penicillamine, colchicine, Relaxin, steroids, andcyclosporine. C-terminal endostatin polypeptides also can be used incombination with immunosuppressive agents. Additionally, the C-terminalendostatin polypeptides can be used with methotrexate, cyclophosphamide,azathioprine, mycophenolate, glitazones, endothelin receptorantagonists, or Fulvestrant (ICI-182,780).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES

Excessive deposition of extra cellular matrix (ECM) components such asfibronectin (FN) and type I collagen (Col1α1) by organ fibroblasts isdefined as fibrosis. Organ fibrosis is the final common pathway for manydiseases that result in end-stage organ failure. However, effectivetherapy for organ fibrosis is still unavailable (see, for example,Bjoraker et al., Am. J. Respir. Crit. Care. Med. 2000; 157:199-20; Vargaand Abraham, J Clin Invest 2007; 117:557-67; Wynn, J Clin Invest 2007;117:524-29). Uncontrollable wound-healing responses, including acute andchronic inflammation, angiogenesis, activation of resident cells, andECM remodeling, are thought to be involved in the pathogenesis offibrosis (Wynn, J Clin Invest 2007; 117:524-29; Kalluri and Sukhatme.Curr Opin Nephrol Hypertens 2000; 9:413-8). TGF-β is the prototypefibrotic cytokine that is increased in fibrotic organs and contributesto the development of fibrosis by stimulating the synthesis of ECMmolecules, activating fibroblasts to α-smooth muscle actin(α-SMA)-expressing myofibroblasts, and downregulating matrixmetalloproteinases (MMPs) (Branton, Microbes Infect 1999; 1:1349-65;Varga and Pasche Nature Reviews Rheumatology 2009; 5:200-6). Despitehigh expectations, a clinical trial of a monoclonal anti-TGF-β antibodyin patients with early SSc failed to show any efficacy (Varga andPasche, Nature Reviews Rheumatology 2009; 5:200-6).

Endostatin is a 20-kDa internal fragment of the carboxy terminus ofcollagen XVIII. It was originally identified in the supernatant of acultured murine hemangioendothelioma cell line with potentantiangiogenic activity (O'Reilly et al., Cell 1997; 88:277-85).Endostatin inhibits endothelial proliferation and tube formation invitro, and tumor growth in vivo (Dhanabal et al., Biochem Biophys ResCommun 1999; 258:345-52). Studies have been conducted to assessendostatin's anti-tumor properties, including clinical trials (Folkman,Exp Cell Res 2006; 312:594-607). The NH₂-terminal domain of endostatinhas been reported as the functional domain responsible for inhibitingangiogenesis (Tjin Than Sjin et al., Cancer Res 2005; 65:3656-63).Although the exact molecular mechanism of its effect remains unclear,integrins, glypicans, flk-1, and nucleolin have been reported asendostatin receptors (Sudhakar et al., Proc Natl Acad Sci USA 2003;100:4766-71; Karumanchi et al., Mol Cell 2001; 7:811-22). Recent studieshave shown that endostatin is increased in serum and/or BALF obtainedfrom IPF and SSc patients with pulmonary fibrosis (for example, Sumi, JClin Lab Anal 2005; 19:146-9).

In the studies discussed herein, the effects of endostatin on fibrosiswere evaluated. The effect of endostatin and endostatin-derived peptideson fibrosis in vitro was assessed using primary human fibroblasts, exvivo using human skin, and in vivo in mice skin treated with TGF-β.Surprisingly, the findings demonstrate that a carboxy-terminal peptideof endostatin has anti-fibrotic activity and provide a novel therapy forfibrotic disorders.

Example 1 Materials and Methods

Reagents and Antibodies. The full-length recombinant human endostatin(rE) was purchased from Sigma-Aldrich (St. Louis, Mo.). Recombinanthuman TGF-β was from R&D Systems Inc. (Minneapolis, Minn.). Mousemonoclonal anti-human fibronectin (FN) antibody, goat polyclonalanti-human type I Collagen αI chain (Col1α1) antibody, and mousemonoclonal anti-human GAPDH antibody were from Santa Cruz Biotechnology(Santa Cruz, Calif.). Mouse monoclonal anti-human α-smooth muscle actin(α-SMA) antibody was from Sigma-Aldrich.

Synthesis of Human Endostatin Peptides. Peptides were synthesized by thesolid-phase on Liberty Microwave Synthesizer (CEM Corporation, Mathews,N.C.) using FMOC synthesis protocol. Briefly, synthesis was performed bystepwise addition of activated amino acids to the solid support (Wangresin and PEG-PS) starting from the carboxy terminus to the aminoterminus. Activation of amino acids was performed by DIPEA/HOBT/TBTUchemistry. At the end of the synthesis, peptides were cleaved off theresin with reagent R (90% TFA, 5% Thioanisole, 3% Ethanedithiol, and 2%Anisole) and subjected to multiple ether extractions. The crude peptideswere analyzed, characterized, and purified by Gel filtration (G-25column), Reversed-Phase High Performance Liquid Chromatography (RP-HPLC,486 and 600E by Waters Corporation). The correct mass was confirmed byMALDI-TOF Mass Spectroscopy (The Voyager-DE STR BiospectrometryWorkstation). Sequences of the peptides are shown in TABLE 1 andcorrespond to amino acids 1-45 (E1); 71-115 (E2); 133-180 (E3), 133-180A(E4) which differs from E3 by the presence of a carboxy-terminal amide.The purity of all peptides was >98%. All peptides were dissolved in DMSOat a concentration 5 mg/ml, and diluted in 1×PBS to 1-20 μg/ml.

Primary Fibroblast Culture. Human primary lung and skin fibroblasts werecultured. The explanted lungs of normal organ donors, patients with SScor IPF, and clinically involved skin of SSc patients, a morphea patientand healthy donors were used for primary fibroblast culture.Approximately 2-cm pieces of peripheral lung and skin were minced andfibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM;Mediatech, Herndon, Va.) supplemented with 10% FBS, penicillin,streptomycin, and anti-mycotic agent, as previously described (Feghaliet al., Arthritis Rheum 1999; 42:1451-7). All the cells were usedbetween passages 3-6.

Western Blot Analysis. Cellular lysates were obtained from culturedfibroblasts as previously described (Pilewski et al., Am J Pathol 2005;166:399-407). Briefly, 2.0×10⁵ primary fibroblasts were cultured in35-mm wells in 0.5% FBS-containing medium supplemented with 10 ng/ml ofhuman recombinant TGF-β or PBS as vehicle control for 24 h, followingwhich 5 μg/ml of human rE, endostatin peptides (E1-E4), or DMSO(vehicle) was added for 48 h. In some experiments, endostatin peptideswere used without TGF-β stimulation. Cellular lysates were analyzed byWestern blot. Signals were detected following incubation withhorseradish peroxidase-conjugated secondary antibody andchemiluminescence (Perkin Elmer Life Sciences, Inc., Boston, Mass.). Theintensity of individual bands with expected molecular sizes wassemi-quantitatively analyzed using the image/J® software available at onthe internet (/rsb.info.nih.gov/ij/index.html), and normalized toindividual GAPDH intensity.

Ex vivo human skin assays. Human abdominal skin was obtained fromcorrective plastic surgery. As previously described (Yasuoka et al., TheOpen Rheumatol J 2008; 2:17-22), subcutaneous fat tissue was removeduniformly and skin tissue was cut into 1.5 cm×1.5 cm sections. Thefollowing were injected intradermally in a total volume of 100 μl 1×PBS:rE alone (1-10 μg/ml), endostatin peptides alone (10 μg/ml), rE orendostatin peptides (1-20 μg/ml) in combination with TGF-β (10 ng/ml),and TGF-β alone (10 ng/ml). In some experiments, human skin was firstinjected with TGF-β for 48 h followed by recombinant endostatin (rE)administration in the same injection site as TGF-β. Independentexperiments were conducted in duplicate or triplicate as indicated inthe figure legends. Explants containing complete epidermal and dermallayers were cultured in an air liquid interface with the epidermal andkeratin layers side up and exposed to air. The culture medium wasreplaced every other day. After 1 or 2 weeks, skin tissue correspondingto an area with 8-mm diameter centered around the injection site washarvested using disposable 8-mm ACUPUNCH® (Acuderm, Inc., Lauderdale,Fla.). Skin tissue was fixed in 10% formalin prior to embedding inparaffin.

In Vivo Mouse Experiments. CB57BL6/J male mice were purchased from TheJackson Laboratory (Bar Harbor, Me.). Human rE (10 μg/ml) or Endostatinpeptides (10 μg/ml) in combination with TGF-β (10 ng/ml), or TGF-β alonewere injected intradermally on the back of mice in a total volume of 100μl 1×PBS. Mice were injected in two different skin sites and sacrificedone week post-injection. Skin surrounding the injection site washarvested and fixed in 10% formalin prior to embedding in paraffin.

Measurement of Skin Dermal Thickness. Six μm sections ofparaffin-embedded human and mouse skin tissues were stained withhematoxylin and eosin (H&E). In some experiments, sections were stainedwith Masson trichrome which identifies collagens. Images were taken on aNikon Eclipse 800 microscope. The thickness of the dermis was measuredin 6 random fields of each section using the image/J® software. Data areshown in arbitrary units.

Tubular formation assay. The ability of endostatin peptide to inhibitangiogenesis was examined in tubular formation assay using MATRIGEL®culture. Human umbilical vein endothelial cells (HUVECs) were maintainedin endothelial cell basal medium-2 (EBM-2; Clonetics, San Diego, Calif.)supplemented with EBM-2 MV SINGLEQUOTS®. HUVECs (5×10⁴) were cultured induplicate on 24-well MATRIGEL® plates (BD Biosciences, San Diego,Calif.) alone, or in the presence of rE or E4 peptide (50 nM) in EBM-2at 37° C. DMSO was used as vehicle control. After 24 hours, images werecaptured using a converted microscope. The degree of cord formation wasquantified by measuring the area occupied by tubes in 6 random fieldsper well. Three independent experiments were performed.

Statistical Analysis. All continuous variables were expressed as themean±standard deviation. Comparisons between 2 groups were tested forstatistical significance using the paired t-test or Mann-Whitney U testas appropriate. Comparison among 3 groups was performed using ANOVAfollowed by Bonferroni's test.

Example 2 Human Endostatin Inhibits FN and Col1α1 Production inTGF-β-Treated Human Primary Lung and Skin Fibroblasts In Vitro

To evaluate whether endostatin modulates production of ECM components infibroblasts, FN and Col1α1 expression was examined in normal human lungfibroblasts by Western blot analysis. Cells were treated with 5 μg/ml rEfor 48 h with or without pre-stimulation with human TGF-β for 24 h. Asshown FIG. 1A, rE dramatically reduced FN and Col1α1 levels in TGF-βpre-treated fibroblasts. To define the functional domain of endostatinthat mediates its inhibitory effect, four different peptides weresynthesized corresponding to different regions of endostatin (TABLE 1).

TABLE 1 Amino acid sequence of human endostatin fragments. E1 (amino acids 1-45 of SEQ ID NO: 2) H-¹HSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGT⁴⁵-OHE2 (amino acids 71-115 of SEQ ID NO: 2) H-⁷¹IVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWP¹¹⁵-OHE3 (SEQ ID NO: 20; amino acids 133-180 of SEQ ID NO: 2) H-¹³³SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMT¹⁸⁰-OHE4 (SEQ ID NO: 21; amino acids 133-180A of SEQ ID NO: 2) H-¹³³SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMT¹⁸⁰-CONH₂ 

As shown in FIGS. 1B and 1C, a fragment from the carboxy terminus ofendostatin (E4) significantly suppressed FN and Col1α1 production inTGF-β treated cells compared with normal lung fibroblasts treated withTGF-β alone (P=0.03, in both comparisons). On the other hand, E1peptide, located in the amino terminal region of endostatin, had noeffect. In addition to healthy fibroblasts, lung fibroblasts obtainedfrom SSc and IPF patients, who had clinical lung fibrosis, were used inparallel assays with similar results (FIGS. 1B and 1C). Havingdemonstrated anti-fibrotic effects of rE and E4 in lung fibroblasts, theeffects of these peptides was examined on skin fibroblasts since skin isa major organ affected by fibrosis in SSc. Primary fibroblasts obtainedfrom the skin of healthy controls, patients with systemic sclerosis(SSc) or localized scleroderma (morphea) were treated with rE or E4.Similarly to lung fibroblasts, rE and E4 reduced TGF-β-induced ECMproduction in dermal fibroblasts. Representative results are shown inFIG. 1D.

Example 3 Endostatin Peptides Reverse the Fibrotic Phenotype of PrimaryLung Fibroblasts from Patients with SSc and IPF

Since it has been shown that TGF-β is upregulated in fibrotic tissue, itwas examined if matrix production in fibrotic lung fibroblasts wasaltered by treatment with endostatin peptide in the absence of TGF-βstimulation. As shown in FIG. 1E left panel, both FN and Col1α1 levelsdecreased in E4-treated fibroblasts. In addition, the same fibroblastswere treated with different concentrations of E4 to identify the optimalanti-fibrotic dose. E4 dose-dependently reduced Col1α1 levels whencompared to vehicle control (FIG. 1E, right panel), but had a modesteffect on FN levels. The reduction in ECM was more modest than thatobserved following TGF-β stimulation. Taken together, the resultsindicate that E4 can reduce baseline production of ECM components infibroblasts from a fibrotic milieu and thus reverse the fibroticphenotype.

Myofibroblasts, activated fibroblasts which express α-SMA, are inducedby TGF-β stimulation and play a central role in fibrosis. Therefore, theeffects of endostatin peptides on α-SMA expression in normal lungfibroblasts was examined. As shown in FIG. 1F, TGF-β stimulation greatlyincreased α-SMA expression. Interestingly, E4, and to a lesser extentE3, decreased TGF-β-induced α-SMA levels suggesting that thecarboxy-terminal region of endostatin can prevent the activation offibroblasts and their transition to a myofibroblastic phenotype.

Example 4 Endostatin Reduces Dermal Thickness and Prevents TGF-β-InducedFibrosis in Human Skin

Cultured human skin explants can be used as an organ model to assess theeffects of fibrogenic factors and for evaluating the efficacy ofinhibitors/therapies to halt the progression of fibrosis and potentiallyreverse it (Yasuoka, The Open Rheumatol J 2008; 2:17-22). To evaluatethe efficacy of endostatin as a potential therapeutic agent forfibrosis, this ex vivo human skin model was used. Since TGF-β is awell-known pro-fibrotic factor that plays a central role in fibrosis,human recombinant TGF-β was first injected intradermally to assess thelevel of fibrosis. As shown in FIG. 2A, TGF-β injection dramaticallyincreased dermal thickness in a dose-dependent manner one weekpost-injection. The fibrotic effect of TGF-β (10 ng/ml) resolved by twoweeks. The baseline effects of rE (1, 5, and 10 μg/ml) or endostatinpeptides (10 μg/ml) were also examined individually. Although rE andE1-4 did not significantly alter dermal thickness, rE, E3, and E4 showeda tendency towards reduction in human dermal thickness (FIGS. 2B and2C). It was determined if rE could inhibit fibrosis in TGF-β-treatedhuman skin. TGF-β and rE were injected simultaneously. One weekpost-administration, rE in combination with TGF-β significantly reduceddermal thickness in a dose-dependent manner (FIG. 3). To assess theeffects of rE on reversing fibrosis, the peptide was injected 2 daysafter TGF-β administration. Similarly to co-treatment, delayed rE alsosignificantly ameliorated TGF-β-induced dermal fibrosis. The findingsindicate that human endostatin can prevent the development andprogression of fibrosis and also reverse TGF-β-induced fibrosis in humanskin.

Example 5 Endostatin Peptides Reduce TGF-β-Induced Fibrosis in HumanSkin Ex Vivo and Reverse Existing Fibrosis

To determine which part of endostatin is responsible for inhibitingTGF-β-induced fibrosis in human skin explants, endostatin peptides (10μg/ml) were administrated in the presence of 10 ng/ml of TGF-β.Representative images are shown in FIG. 4A. E3 and E4 significantlyabolished the development of fibrosis as measured by dermal thicknesswhen compared to TGF-β alone (P=0.04, 0.01, respectively; FIG. 4). Thedermal thickness of skin explants injected with different concentrationsof E1 or E4 in combination with TGF-β was examined. As shown in FIG. 5,unlike E1, E4 at concentrations of 5-20 μg/ml clearly amelioratedTGF-β-induced skin fibrosis, indicating that the C-terminus ofendostatin can suppress fibrosis (see FIG. 17).

Example 6 Endostatin Peptides Reduce TGF-β-Induced Fibrosis In Vivo inMouse Skin

The anti-fibrotic effect of endostatin peptides was further assessed invivo. rE and endostatin peptides in combination with TGF-β were injectedin the skin of mice. One week post-injection, mice appeared healthy andshowed no signs of distress. As shown in FIG. 6, human TGF-β stronglyincreased dermal thickness in mouse skin (P=0.004). Peptides E3 and E4from the carboxy terminus of human endostatin peptide prevented dermalfibrosis induced by TGF-β (P=0.01, 0.007, respectively). In addition, E2significantly reduced dermal thickness (P=0.03). E1, a peptidecorresponding to the amino terminus of endostatin did not alterTGF-β-induced dermal fibrosis. These results confirmed those obtained inour human skin model and emphasize the importance of the C-terminaldomain of endostatin in preventing TGF-β-induced fibrosis in vivo and exvivo.

Example 7 The C-Terminal Peptide of Endostatin has ModestAnti-Angiogenic Activity

The anti-angiogenic effect of endostatin has been attributed to itsamino terminal domain (Tjin Tham Sjin et al., Cancer Res 2005;65:3656-63). To evaluate the anti-angiogenic capacity of the carboxyterminal regions of endostatin, the effect of E4 on in vitro tubularformation was examined using Matrigel. As shown in FIG. 7, the capacityof rE to inhibit tubular structure formation by HUVECs was significant,confirming previous reports. On the other hand, the ability of E4 tosuppress angiogenesis was modest, suggesting that the region ofendostatin corresponding to E4 does not significantly contribute to itsanti-angiogenic activity.

Thus, E4, a peptide corresponding to the carboxy terminal region ofendostatin, ameliorates TGF-β-induced fibrosis and even reverses it. E4suppressed TGF-β-induced ECM production and downregulated α-SMA levelsin primary lung and skin fibroblasts. In vivo and ex vivo analysesrevealed that E4 impedes the increase of skin dermal thickness triggeredby TGF-β. Furthermore, the anti-angiogenic capacity of E4 was lowcompared to that of rE. Taken together, the findings suggest that thedomains of endostatin responsible for its anti-fibrotic andanti-angiogenic capacity are distinct. Other endostatin peptides (forexample, E2 and E3) are shown to have anti-fibrotic activity.

The anti-angiogenic activity of endostatin has been the focus ofnumerous investigations directed at the development of anti-tumortherapy. Recently, elevated serum and BALF levels of endostatin infibrotic disorders such as idiopathic pulmonary fibrosis (IPF) andsystemic sclerosis (SSc) were reported. Endostatin levels wererelatively increased in IPF patients with severe respiratory dysfunctionand in SSc patients with pulmonary fibrosis, severe skin fibrosis, andwith cutaneous scars, compared to patients without those clinicalmanifestations (Sumi J Clin Lab Anal 2005; 19:146-9; Richter et al.,Thorax 2009; 64:156-61). In addition, collagen XVIII expression wasincreased in cultured dermal fibroblasts of SSc patients (Tan et al.,Arthritis Rheum 2005; 52:865-76) and in whole lung extracts of patientswith IPF (Yang et al., Am J Respir Crit Care Med 2007; 175:45-54). Inthis regard, since endostatin is a proteolytic product of collagen XVIIIcleaved by several proteases including MMPs and cathepsin L (Wen et al.,Cancer Res 1999; 59:6052-6; Felbor, EMBO J 2000; 19:1187-94), and sinceMMPs are also upregulated in SSc and IPF (Richter et. al., Thorax 2009;64:156-61, Toubi et al., Clin Exp Rheumatol 2002; 20:221-4), theobservations that cleaved endostatin levels are elevated in thosepatients is plausible. However, it is unclear how endostatin may beinvolved in the pathogenesis of fibrosis.

Without being bound by theory, increased endostatin in fibrotic tissuesmay constitute a negative feedback regulatory loop which, althoughunsuccessful, is directed at halting the progression of fibrosis. Sinceendostatin was originally identified in aberrant “angiogenic”endothelial cancer cells as a product that likely controls/inhibits its“angiogenic” capacity (O'Reilly et al., Cell 1997; 88:277-85), it isplausible that endostatin in fibrosis serves a similar regulatoryfunction.

Reduced connective tissue but normal vessel density has been reported inrecombinant endostatin-treated mouse skin using a wound healing model(Bloch et al., FASEB J 2000; 14:2373-6). Further, a peptide from theN-terminal region of endostatin prevented the progression of peritonealsclerosis in a mouse model (Tanabe et al., Kidney Int 2007; 71:227-38);the peptide under investigation corresponded to the N-terminus ofendostatin encompassing amino acids 1-27.

In contrast, the C-terminal region of endostatin, but not theN-terminus, is shown herein to be responsible for its anti-fibroticeffects. In fact, the peptide corresponding to the N-terminal domain ofendostatin contributed to the fibrotic phenotype in some of the assays.Studies directed at defining the specific amino acid sequenceresponsible for endostatin's anti-angiogenic capacity (Richter et al.,Thorax 2009; 64:156-61; Cattaneo et al., Exp Cell Res 2003; 283:230-6;Xu et al., Curr Protein Pept Sci 2008; 9:275-83) have shown that theentire angio-suppressive activity of endostatin was located in a27-amino-acid peptide in the N-terminal domain (Richter et al., Thorax2009; 64:156-61). Thus, the functional domain of endostatin thatmediates its anti-fibrotic activity is different from that responsiblefor its anti-angiogenic capacity, implying different mechanisms forinhibiting angiogenesis and fibrosis. The anti-fibrotic C-terminalendostatin polypeptides disclosed herein are therefore capable ofselectively inhibiting fibrosis without inhibiting angiogenesis. TheC-terminal endostatin polypeptides can be used to more specifically andselectively target unwanted fibrosis without interfering withangiogenesis that may impact a desired therapeutic outcome.

The C-terminal endostatin polypeptide also reduces α-SMA expression inTGF-β-treated fibroblasts. In addition, the matrix reducing effects ofE4 on normal fibroblasts was modest compared to that in fibroticfibroblasts. This suggests that the therapeutic effect of endostatinC-terminal peptide in fibrosis could be due, in part, to hindrance offibroblast activation by TGF-β and other fibrosis promoting growthfactors.

In 2005, ENDSTAR®, a recombinant human endostatin purified from E. colicontaining an additional nine-amino acid sequence produced as aHis-tagged protein was approved for the treatment of non-small-cell lungcancer in China (Sun et al., J Clin Oncol 2005 (ASCO Annual meetingproceedings); 23:7138). Despite its effectiveness, the treatment hadseveral disadvantages including a requirement for high doses, theprotein's short half-life, poor stability and easy inactivation (see,for example, Crystal, Nat Biotechnol 1999; 17:336-7; Hu et al., ActaPharmacol Sin 2008; 29:1357-69). The small synthetic peptides disclosedherein could overcome these obstacles. E4 significantly inhibitedfibrosis compared to rE and even E3 in vitro, in vivo, and ex vivo. Inaddition, E4 had minimal anti-angiogenic activity compared to rE,confirming that the anti-angiogenic activity of endostatin resides inits N-terminal domain. The only difference between E3 and E4 was thepresence of an amide-bond in the C-terminus of E4. Without being boundby theory, this amide renders the peptide more resistant to carboxydegradation by carboxypeptidases or other degrading molecules, thusstabilizing the peptide and likely maintaining its biological activity(Yang et. al. Am J Respir Crit Care Med 2007; 175:45-54).

Unfortunately, there are no effective therapies for organ fibrosis. TheC-terminal domain of endostatin, corresponding to amino acid sequence133-180 with amide-bond formation, suppressed ECM production by primaryskin and lung fibroblasts and ameliorated dermal fibrosis induced byTGF-β in vivo and ex vivo in human skin. The findings presented hereindemonstrate that E4 could be used for the treatment of fibroticdisorders, including IPF, SSc, morphea, as well as Graft-versus-hostdisease, keloid and hypertrophic scar, and other organ fibrosis such assubepithelial fibrosis in asthma.

Example 8 Confirmation of the Efficacy of E4

E4, a peptide representing the carboxy terminus of human endostatin, canattenuate fibrosis triggered by multiple fibrogenic factors. Theanti-fibrotic effects of E4 can be detected whether administeredconcomitantly with or following the fibrogenic trigger. The efficacy ofE4 was confirmed in four pre-clinical models of fibrosis: a)bleomycin-induced dermal fibrosis in vivo in mouse skin, b) TGF-βinduced dermal fibrosis in mouse skin, and c) bleomycin-inducedpulmonary fibrosis. E4 peptide or a control peptide (E1; representingthe amino terminal region of endostatin) were administered at the sametime as TGF-β or bleomycin or 3-4 days following TGF-β or bleomycin.Mice were sacrificed one and two weeks after TGF-β-initiation of dermalfibrosis, and two and three weeks after bleomycin-induced pulmonaryfibrosis. Two different modes of administration of the E4 peptide werealso tested. It was confirmed that intraperitoneal and intratrachealadministration was effective. The amount of E4 that was administered was10 μg/ml in a total volume of 100 μl for skin and IP injections and 50μl for IT administration.

For these studies, fibrosis was assessed by measurement of dermalthickness on H&E skin sections (skin), assessment of collagen levels byMasson Trichrome staining (skin and lung), and measurement of collagenlevels by Sircol assay (lung). Furthermore, to confirm the mechanism bywhich E4 exerts its anti-fibrotic effects, the production ofextra-cellular matrix (ECM) components, the levels of enzymes thatpromote matrix stabilization and thus accumulation and levels of thosethat degrade ECM components, and levels of transcription factorsdownstream of the pro-fibrotic triggers were evaluated. Results wereassessed using the unpaired t test and the 3-way ANOVA (for the ID1data).

Results

E4 caused a significant attenuation of bleomycin induced dermal fibrosiseven with a single administration of E4 (FIG. 8). E4 caused asignificant decrease of TGF-β induced dermal fibrosis on day 7. Thus E4prevents (FIG. 8) and reverses (FIG. 9) dermal fibrosis triggered byTGF-β.

E4 administered concomitantly with bleomycin or three days followingbleomycin caused a marked reduction in fibrosis and Masson Trichromestaining (see FIG. 9 and FIG. 10). E4 peptide given three days afterbleomycin significantly reduced collagen levels in mouse lungs (FIG.10B).

E4 caused a statistically significant reduction in both TGF-β andbleomycin induced skin (FIG. 8) and lung fibrosis (FIG. 10) regardlessof the mode of administration. Intraperitoneal and intratrachealadministration of E4 were both effective in blocking dermal andpulmonary fibrosis. For example, E4 caused a significant attenuation ofbleomycin induced lung fibrosis on day 21 whether administeredintraperitoneally or intratracheally (FIG. 11). Thus E4 is effective atreducing fibrosis irrespective of the administration mode.

The results also evidenced that E4 exerts its anti-fibrotic effects viamultiple pathways. E4 reduces levels of lysyl oxidase (LOX), and enzymeresponsible for the cross-linking of collagen, elastin, and otherextracellular matrix (ECM) molecules and thus the stabilization of theECM. E4 can make collagen less stable and more susceptible toproteolytic degradation. FIG. 12 shows lung sections of mice treatedwith bleomycin with or without E4.

E4-mediated reduction of LOX was detected also was detected in vitro.Normal lung fibroblasts in passage 4 were treated with vehicle, E4,TGF-β, or TGF-β followed 30 minutes later by E4 (FIG. 13). Mediaconditioned by the fibroblasts were analyzed using Western blot analysisafter 48 hrs. Treatment with E4 significantly reduced the level of LOX.Similar results were obtained when LOX mRNA levels were examined byreal-time PCR.

E4 also promotes the degradation of ECM components via induction andactivation of matrix metalloprotease (MMP-2), an enzyme that degradesseveral ECM molecules including fibronectin and native and denaturedcollagens (FIG. 14). In addition, E4 increases levels of inhibitor ofdifferentiation (ID)-1, a transcription factor that inhibits TGF-βeffects (see FIG. 15). It was determined in a Western blot analysis thatE4 reduces the levels of the master switch transcription factor, Egr-1(see FIG. 16) in primary human lung fibroblasts, treated and harvestedafter 24 hours. The reduction of Egr-1 levels parallels a reduction incollagen, SMA and fibronectin. Egr-1 is known to mediate the effects ofseveral fibrotic agents (including TGF-β and bleomycin).

Thus, E4 exerted significant anti-fibrotic effects. This peptidesignificantly attenuates the fibrogenic effects of TGF-β and bleomycinwhether administered simultaneously with these fibrotic triggers or afew days following the initiation of fibrosis, suggesting that E4, andother C-terminal endostatin polypeptides is also effective at reversingestablished fibrosis. The anti-fibrotic effects of E4 were noted whetherit was administered intratracheally or intraperitoneally to mice inwhich pulmonary fibrosis was induced by bleomycin and dermal fibrosiswas induced by TGF-β. Furthermore, E4 exerted its anti-fibrotic effectsvia multiple pathways that included destabilization of ECM throughreduction of LOX and thus decreased ECM crosslinking, induction of ECMdegradation via activation of MMP-2, suppression of Egr-1 levels, andincreased transcription factor ID-1.

Thus, several in vitro assays and four in vivo and ex vivo pre-clinicalmodels of fibrosis suggest that C-terminal endostatin polypeptides, asexemplified by E4, are an effective anti-fibrotic peptide that can blockand reverse fibrosis in two organs, lung and skin. These anti-fibroticeffects as well as the lack of anti-angiogenic effects characteristic ofendostatin render E4 an attractive therapeutic peptide for organfibrosis.

Example 9 Production of an Orally Active Anti-Fibrotic Protein in Plants

Studies were conducted to evaluate the expression, purification, andoral delivery of the E3-6His-KDEL, E3-Fc (C67A), and ExpandedE3-6His-KDEL peptides shown in TABLE 2.

Methods

Peptides were expressed in plants using the IBIOLAUNCH™ gene expressionplatform. C57BL/6 mice were given bleomycin intratracheally inconjunction with END-55 (also referred to herein as E3-Fc (C67A) andCFB03) intravenous (IV) administration, or followed by IV administrationof END-55 eight days after bleomycin. The mice were sacrificed at day 12and lung samples were taken for both hematoxylin and eosin (H&E)staining and hydroxyproline assay.

Results

E3 peptides were expressed in plants using the IBIOLAUNCH™ platform.Untagged E3 peptides were purified based upon differential solubility.See FIG. 18. Each of E3 variant peptides (E3-6His-KDEL, ExpandedE3-6His-KDEL (C67A), and E3-Fc (C67A)) was successfully produced andpurified. See FIG. 19.

Pulmonary fibrosis was induced in mice using bleomycin. Animals weretreated orally with E3 variants or phosphate buffered saline (PBS). Lunghistology was examined, showing that orally delivered E3 variants(E3-6His-KDEL and Expanded E3-6His-KDEL) prevented bleomycin-inducedpulmonary fibrosis. See FIG. 20.

For the experiments shown in FIGS. 21 and 22, pulmonary fibrosis wasagain induced in mice using bleomycin. Controls were administered PBS,while experimental samples also had END55 (also referred to as E3-Fc(C67A)) administered orally or intravenously. Hydroxyproline content wasmeasured in the lung.

Compared to mice treated with bleomycin only, mice treated with END-55at 100 μg or 200 μg had reduced pulmonary fibrosis, as assessed usinghydroxyproline assays (FIG. 21). Intravenously administered END-55 ateither dose appeared to have similar efficacy to orally administeredEND-55 at 50 μg. END-55 administered eight days after bleomycintreatment (both orally and intravenously) also appeared to reducefibrosis, although only three mice were treated in each of these groups.These studies thus indicated that oral and IV delivery of END55prevented bleomycin-induced pulmonary fibrosis, and that IV dosing ofthe polypeptide was comparable to oral administration. See, FIGS. 21 and22. In addition, END-55 ameliorated bleomycin-induced pulmonary fibrosisin vivo whether administered concomitantly or after a fibrotic trigger(represented as d8; FIG. 21). Therefore, IV administration of END55 hasthe potential to reverse lung fibrosis.

TABLE 2 SEQ ID  Construct Sequence NO: Description END16MGKMASLFATFLVVLVSLSLASESSASYC 15 E3-6His-KDEL: secretory leader peptideETWRTEAPSATGQASSLLGGRLLGQSAASC (bold); tagged with 6X His and KDELHHAYIVLCIENSFMTHHHHHHKDEL sequence (italics) END55MGKMASLFATFLVVLVSLSLASESSASYC 16 E3-Fc (C67A): secretory leader peptideETWRTEAPSATGQASSLLGGRLLGQSAASC (bold); third cysteine changed to alanine

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVV to human immunoglobulin IgGl FcVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE domain (italics)QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK END56

17

Example 10 Ig Molecules Fused to E3

Various Fc domain fusions to E3 were expressed (in addition toE3-Fc(IgG1)). Sequences were as follows:

E3_C67A-Fc_IgG2: (SEQ ID NO: 22)MGKMASLFATFLVVLVSLSLASESSASYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLAIENSFMTERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKE3_C67A-Fc_IgG3: (SEQ ID NO: 23) MGKMASLFATFLVVLVSLSLASESSASYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLAIENSFMTELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNR FTQKSLSLSPGKE3_C67A-Fc_IgG4:  (SEQ ID NO: 24)MGKMASLFATFLVVLVSLSLASESSASYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLAIENSFMTESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGKE3 _C67A-Fc_IgA1:  (SEQ ID NO: 25)MGKMASLFATFLVVLVSLSLASESSASYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLAIENSFMTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY E3_C67A-Fc_IgA2:  (SEQ ID NO: 26)MGKMASLFATFLVVLVSLSLASESSASYCETWKTEAFSATGQASSLLGGRLLGQSAASCHHAYIVLAIENSFMTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY E3_C67A-Fc_IgM:  (SEQ ID NO: 27)MGKMASLFATFLVVLVSLSLASESSASYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLAIENSFMTVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY J-Chain_PVX_sgp36:  (SEQ ID NO: 28)MGKMASLFATFLVVLVSLSLASESSAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD IgGI_Fc-E3_C67A (C-terminal fusion): (SEQ ID NO: 29) MGKMASLFATFLVVLVSLSLASESSAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVICFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLAIENSFMT

As shown in FIG. 23, fusion of E3 to the Fc region of IgG2 demonstratedpoor expression and was insufficient for purification, fusion to -IgG3was soluble and purified, fusion to -IgG4 was soluble and purified,fusion to -IgA1(±J-chain) was insoluble, fusion to -IgA2(±J-chain) wasinsoluble, and fusion to -IgM (±J-chain) demonstrated poor expressionand was insufficient for purification, while Fc-E3 was soluble andpurified.

For IgG Fc expression, clarified plant lysates were run on a Westernblot (Genway chicken E3 antibody). Fusions included E3 (C67A) linked toeither the N-terminus of the hinge+Fc domains of IgG2, IgG3, or IgG4, orto the C-terminus of the hinge+Fc domain of IgG1. The IgG2 fusiondemonstrated low expression, while the IgG3 and IgG4 fusionsdemonstrated moderate expression. The E3-IgG1 fusion expressed well. Thenon-reducing samples all appeared to form large structures, althoughperhaps not as large as the END-55 (iBioCFB03) HMW multimers (FIG. 24).

For IgA/M expression, clarified plant lysates were ground in eitherdenaturing (total) or non-denaturing buffer (soluble) and run on aWestern blot (Genway chicken E3 antibody). Fusions contained E3 (C67A)linked to the N-terminus of the hinge+Fc domains of IgA1, IgA2, or IgM.The IgA1 and IgA2 fusions were insoluble in normal grind buffer. The IgMfusion had very low expression. J-chain co-expression had no obviouseffect. See FIG. 25

Example 11 iBio-CFB03: High Molecular Weight Multimer

iBio-CFB03 (also referred to herein as END55 or E3-Fc (C67A)) is a highmolecular weight multimer comprised of multiple subunits of a 306 aminoacid-length fusion protein, with a 48 amino acid fragment derived fromthe C terminus of Collagen XVIII (sequence of E3), containing an addedsecretory leader peptide [bold; 26 amino acids; cleaved during secretionof the molecule into the endoplasmic reticulum (ER) by ER-localizedsignal peptidases] and fused to human immunoglobulin IgG1 Fc domain(italics; 232) as shown in FIG. 26. This fusion protein also contains acysteine to alanine mutation at position 67 (underlined and bolded) andone glycosylation site (italics and underlined). The predicted molecularweight of the non-glycosylated E3-Fc is 31,153 Da.

Chemistry, Manufacturing and Controls

iBio-CFB03 is a multimeric protein approximately 1.2 mega-Daltons (MDa)with a repeating monomer with two major glycoforms between 31 and 33kDa. The multimer is soluble and stable when stored at 4° C. inphosphate buffered saline (PBS).

Method of Manufacture

iBio-CFB03 Drug Substance was manufactured at Caliber Biotherapeutics,LLC (8800 HSC Pkwy, Bryan, Tex. 77807) using the IBIOLAUNCH™ platformthat utilizes a transient plant-based expression system allowingagricultural scaling of the upstream production, followed by traditionaldownstream purification and formulation processes. An overview of themanufacturing processes is presented in FIG. 27.

Summary of Drug Substance Manufacturing Process Generation of MasterCell Bank (MCB) and Working Cell Bank (WCB)

Codon Optimized Sequence

The genetic sequence of the endostatin E3 peptide (amino acid 1,466 to1,513 of the human collagen alpha-1 (XVIII) chain preproprotein, GenBankNP_085059.02) was modified by mutating the last cysteine in the E3peptide to alanine using Strand Overlap Extension (SOE) with mutantoligos. The resulting E3 peptide mutant C67A sequence was fused to thesignal peptide of the Nicotiana plumbaginifolia extensin gene (GenBankAAA34073.1) and to the human immunoglobulin G1 Fc region (amino acid 216to 447, GenBank: CAC20454) by SOE to form the candidate gene iBio-CFB03(TABLE 3; FIG. 26). The iBio-CFB03 gene was codon optimized for plantexpression using the Nicotiana tabacum codon usage table and cloned intothe IBIOLAUNCH™ tobacco mosaic virus-based vector pGR-D4 betweenrestriction sites PacI and XhoI to generate vector pSM042 (FIG. 28).

TABLE 3 iBio-CFB03 Sequence Description Amino acid position Sequencedescription 1-26 Nicotiana plumbaginifolia extensin signal peptide27-74  Endostatin E3 peptide mutant C67A 75-89  Hinge region of thehuman IgG1 heavy chain 90-306 Fc region of the human IgG1 heavy chainClone into Agrobacterium Vector Strain GV3101

Expression vector pSM042 was then mobilized into Agrobacteriumtumefaciens strain GV3101 together with the helper plasmid, pSOUP, byelectroporation (GenBank: EU048870.1) (Hellens et al., Plant Mol Biol.2000; 42(6):819-832). pSoup encodes RepA, which acts in trans upon thepSa Ori sequence contained in pGreen to permit its replication inAgrobacterium. Agrobacterium clones were selected on Luria-Bertani (LB)agar plate against 50 mg/L kanamycin, 25 mg/L rifampicin and 10 mg/Ltetracycline. Kanamycin resistance was provided by the pGR-D4 expressionvector, rifampicin resistance was provided by the GV3101 Agrobacteriumstrain and tetracycline resistance was provided by the helper vectorpSOUP. After 3 days of incubation at 28° C., single colonies were pickedto inoculate liquid LB media (supplemented with antibiotics as describedabove) to amplify each bacterial culture in a shaker incubator at 28°C., 225 rpm. Each clone was identified by the polymerase chain reaction(PCR) and specific inserts were confirmed by sequencing using specificprimers binding upstream of the insert in the pGR-D4 expression vector.Once confirmed, initial Agrobacteria MCB and WCB were generated andstored at −70° C. in two separate freezers.

iBio-CFB03 Expression

Step 1—Tray Assembly, Seed Trays

Aluminum plant growing trays, nominally 4 feet by 3 feet in size, wereused. Styrofoam plug trays containing 240 plugs of rockwool were seededin an automated vacuum needle seeder. Four plug trays (960 plants) wereplaced in each of the aluminum germination trays and conveyed to thegermination room.

Step 2—Plant Germination Growth

The plants were germinated for 3 weeks at 27° C. in vertical racksequipped with LED lighting with spectrally correct light of regulatedintensity to optimize photosynthesis and optimum plant growth. Dripirrigation was employed to deliver a modified Hoagland's solution ofrequired minerals for hydroponic growth. No soil or other materials wereused to support plant growth.

Step 3—Transplant to Lower Density Trays

At 3 weeks of growth, the plugs containing the germinated plants weremechanically transplanted to 4 feet by 4 feet growing trays that onlyhad 320 plant positions per tray to allow for the expansion of foliage.The plants were grown for another 2 weeks at 25° C. Each plant wasapproximately 8-10 grams of leaf biomass at this stage.

Step 4—Agrobacterium Growth

The selected Agrobacterium clone was grown from a working cell bank inculture flasks containing LB medium supplemented with 50 mg/L kanamycin,25 mg/L rifampicin and 10 mg/L tetracycline at 28° C. with agitation at225 rpm. Cultures reaching an OD_(600 nm) of ˜1.5 were collected anddiluted 50 fold in reverse osmosis-purified water containing 2 mM MESbuffer, pH 5.6.

Step 5—Vacuum Agroinfiltration of Vector Into Plants

After induction of Agrobacteria for one hour, 5 week old plants werevacuum infiltrated at 23 inch Hg at the gauge. Prior to vacuuminfiltration, Nicotiana benthamiana plants were grown hydroponicallyunder proprietary red/blue LED light for five weeks at ˜27° C. withrelative humidity of ˜50%. Agro-infiltrated plants were incubated underconstant LED light at ˜25° C. with relative humidity of ˜50%.

Step 6—Post-Infiltration Growth

The post infiltration room hydroponic growth conditions maintained theplants at 25° C. under a regulated light regime. After six dayspost-infiltration, the plants were automatically conveyed to the harvestarea for extraction.

iBio-CFB03—Purification Process

Step 7—Plant Harvest—Homogenization and Centrifugal Clarification

Nicotiana benthamiana biomass expressing iBio-CFB03 (E3-Fc) wasmechanically homogenized with a Waring heavy duty laboratory gradestainless steel blender for one minute in aqueous acidified (pH 4.8-5.2)extraction buffer (50 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA, 60 mMascorbic acid, and 1 mM PMSF). Biomass was partially clarified bycentrifugation followed by pH adjustment to 6.2 with 1 N NaOH.

Step 8—Depth Filtration

The extract was further clarified by depth filtration (nominal retentionrating 1.2/0.2 μm).

Step 9—Ultrafiltration

Clarified extract was applied to a hollow fiber tangential flowfiltration (TFF) module containing 1 mm fibers with 750 kDa molecularweight cutoff (MWCO) pore size. Extract was filtered through the 750 kDaMWCO with a shear rate of 6,000-8,000 see and a trans-membrane pressureof 5-7 psi.

Purification of iBio-CFB03 was initially assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). As can be seen inFIG. 29, iBio-CFB03 purified by the above procedure failed to enter thegel in an undenatured state (without heat treatment andβ-mercaptoethanol; lane 6). A sample of iBio-CFB03 purified by Protein Aaffinity chromatography (lane 4) behaved similarly, as did a controlreagent (END-81), a peptide of the same amino acid composition as E3 butwith a scrambled primary sequence, fused to IgG1-Fc (lane 5), Theseresults suggest that iBio-CFB03 naturally occurs as a high molecularweight array when produced in plants, that this behavior is not due tothe purification method, and indeed may be intrinsic to Fc fusionproteins having a similar non-polar N terminal sequence. Whenelectrophoresed under common SDS-PAGE denaturing condition (heat plusβ-mercaptoethanol), the majority of purified iBio-CFB03 (lane 3)resolves into two bands of apparent molecular weight of ˜35 kDa and ˜32kDa. Similar results are obtained for iBio-CFB03 purified by Protein Aaffinity chromatography (lane 1) and the END-81 control reagent (lane2). These results indicate that the dual band pattern is not due to thepurification method or primary sequence of the E3-related portion of thefusion protein, and may be due to differences in the glycosylationpattern of the Fc fusion partner. Additionally, bands with apparentmolecular weights consistent with iBio-CFB03 dimers (˜70 kDa), trimers(˜110 kDa), and tetramers (>150 kDa) are also observed in denaturedsamples, and appear without regard to the purification method as well asin the control reagent. These additional bands indicate that the highmolecular weight arrays are extraordinarily stable and resist commonelectrophoresis denaturing conditions, and may be an additionalattribute of fusions of a hydrophobic peptide to human Fc. Due to thesebehaviors, SDS-PAGE is not a preferred method for characterizingiBio-CFB03.

Purity of the TFF retentate was evaluated with size exclusionchromatography (SEC) and compared to non-infiltrated retentate (FIG.30). Under native non-reducing conditions, the SEC chromatogram revealsa single iBio-CFB03 peak at a retention time of 6 minutes (estimated tocorrespond to a molecular weight of 1.2 MDa) that is completely absentin non-infiltrated biomass. The retentate is largely comprised ofiBio-CFB03 product, with plant host cell proteins contributing theremainder. The product is soluble in PBS and saline at a level of 50mg/mL.

Step 10—Cation Exchange Chromatography

TFF retentate was then prepared for cation exchange (CEX) chromatographyby filtration (nominal retention rating 1.2/0.2 μm), pH adjustment to5.3-5.7 with 1 M acetic acid and conductivity adjustment to 4-6 mS/cm byaddition of ultrapure water. During CEX chromatography, remainingimpurities were removed by washing with up to 250 mM sodium chloride(FIG. 31 CEX Wash; FIG. 32 lanes 3-5 and 9, FIG. 33 top) and iBio-CFB03was eluted with 550 mM sodium chloride at ≥95% purity (FIG. 31 CEXElution; FIG. 32 lanes 6 and 10; FIG. 33 bottom).

SDS-PAGE was used to assess contents of the CEX fractions (FIG. 32).Samples in lanes 2-7 were heated at 70° C. for 10 min in the presence of266 mM β-mercaptoethanol prior to loading. Samples in lanes 9 and 10 didnot contain β-mercaptoethanol and were not heated prior toelectrophoresis. Lanes 3-5 correspond to wash fractions 1A1, 1A4 and 1A9(FIG. 29) containing 100, 150 and 250 mM NaCl, respectively. Lane 6displays elution fraction 1B2 (550 mM NaCl) and shows monomer (˜35 kDa),dimer (˜70 kDa),trimer (˜110 kDa) and tetramer (˜150 kDa) species,observable when the iBio-CFB03 product is exposed to reducing agents(e.g., heat and β-mercaptoethanol). When elution fraction 1B2 is loadedin the absence of β-mercaptoethanol and not heated prior toelectrophoresis (lane 10) the HMW iBio-CFB-03 multimer can be visualizedat the origin of the gel.

Step 11—Diafiltration Buffer Exchange and Sterile Filtration

iBio-CFB03 CEX elution pool material was then concentrated up to 12.5mg/mL and buffer was exchanged to phosphate buffered saline by TFF usinga 100 kDa MWCO TFF (stabilized cellulose) at a transmembrane pressure of2-4 psi.

Step 12—Packaging of Bulk Drug Substance

The bulk drug substance is sterile filtered and packaged in a sterilebag within a biosafety cabinet. The bulk product is transferred to aselected fill facility for final fill.

Characterization of iBio-CFB03SE-HPLC Analysis of iBio-CFB03

iBio-CFB03 samples were analyzed by SE-HPLC to assess the purity of theHMW multimer. SE-HPLC analysis was performed on a TSKgel G3000SW xL, 7.8mm×30 cm, 5 μm column using 1100 Series HPLC system with UV detection.Mobile phase for SE-HPLC consisted of 50 mM sodium phosphate (monobasic,monohydrate), sodium phosphate (dibasic, anhydrate) and 0.3 M sodiumchloride, pH 7.0. Prior to sample analysis, protein mix standard wasprepared and 20 μL was injected onto the column. SE-HPLC protein mixstandard consisting of thyroglobulin (0.5 mg/mL), BSA (1 mg/mL),ovalbumin (1 mg/mL), α-lactalbumin (1 mg/mL) and aprotinin (0.4 mg/mL)was used to determine the molecular weight of the sample chromatographicpeaks. Mobile phase buffer was used for blank injections. The column wasequilibrated with the mobile phase before sample analysis. Samples wereseparated on the column at a flow rate of 1.0 mL/min for a total runtime of 15 min.

Data analysis was performed with ChemStation Data Analysis software(Agilent Technologies, A.01.04 025). Absorbances were recorded at 220 nmand 280 nm. The UV signal of each sample was integrated and the relativepercent abundance of each peak detected was determined. All peaks with apercent relative abundance >0.1% were considered for quantification.

SE-HPLC analysis of iBio-CFB03 under non-reduced conditions displayed amajor peak at 6 min which clearly overlays with the dimer ofthyroglobulin peak (1.2 MDa) from the protein mix standard as shown inFIG. 34, indicating that iBio-CFB03 exists as a higher-order proteinstructure. Another small peak at 10.2 min was also observed whichcorresponds to approximately 50 kDa peak when overlaid with the proteinmix standard. The two small peaks at 12.6 min and 12.9 min were notdetected at 280 nm and were considered non-protein impurities. The peaksat 6 min and 10.2 min retention times were fraction collected separatelyand subject to tryptic digestion to determine their identity usingMALDI-TOF MS.

MALDI-TOF MS Analysis of iBio-CFB03

iBio-CFB03 samples were analyzed by MALDI-TOF-MS under non-reduced andreduced conditions to obtain the molecular weight of the HMW multimerfollowed by tryptic digestion for sequence confirmation. For reducedanalysis, the sample was treated with 0.5 M BME and heated at 70° C. for10 min. Cyano-4-hydroxy cinnamic acid (α-CHCA) (Sigma) and sinapinicacid matrices were used for tryptic peptides and molecular weightdetermination respectively, using MALDI-TOF MS. The sinapinic acidmatrix solution was prepared at 10 mg/mL in 30:70 acetonitrile(ACN):0.1% trifluoroacetic acid (TFA) and the α-CHCA matrix was preparedat 10 mg/mL in 1:1:1 Ethanol: ACN: 0.1% TFA. The samples were then mixedat a 1:9 ratio in a 0.5 mL Eppendorf tube and 1.5 μL of mixture wasspotted on a MALDI plate for analysis.

Samples were allowed to air dry completely prior to analysis as theyco-crystallize with the matrix for approximately 2-3 min. The plate wasthen loaded into the MALDI-TOF mass spectrometer. Acquisitions weremanual for peptide analysis, and the instrument mode was set to“Reflector”, “Delayed”, and “Positive” and the accelerating voltage andgrid were set to 20 kV and between 66-74%, respectively with a delaytime of ˜125 nsec. Shots/Spectrum was set to 150 with a mass range of500-4,000 Da and a low mass gate of 400 Da. α-CHCA matrix was selectedto acquire the calibration and data file spectra. The ion source andmirror pressures were always less than 5.0×10⁻⁷ and 1.2×10⁻⁷ torr,respectively before acquisition was initiated. The initial laser powerwas set to 1250 for calibration and adjusted as necessary duringspectrum acquisition. A two-point mass calibration curve was generatedusing the ACTH (1-17 fragment at 2,093.0867 Da) resolved mass peak andthe Bradykinin (2-9 fragment at 904.4681 Da) mass peak in the standardtest mix and applied to the protein sample analysis as detailed in thestandard operating procedures (SOP). The calibration file was tested byre-acquiring the test mixture spectrum and the file was rejected andre-acquired if the monoisotopic mass for either peptide was >±0.2 Da.The monoisotopic mass for each peptide was used in the calibration.

Similarly, for molecular weight analysis, the instrument mode was set to“Linear”, “Delayed” and “Positive” mode with accelerating voltage 25 kV,grid set to 66-74% and delay time of 200 nsec. Shots/Spectrum was set to200 with a mass range of 4,500-200,000 Da and a low mass gate of 4,000Da. Sinapinic acid matrix was selected to acquire the calibration anddata file spectra. The initial laser power was set to 1800 forcalibration and adjusted as necessary during spectrum acquisition. A onepoint calibration curve was generated using Myoglobin (11,652.52 Da)average mass peak.

The MALDI spectrum of iBio-CFB03 under reduced conditions displayed m/z31,166.94 [M+H]⁺ and m/z 33,068.88 [M+H]⁺ representing non-glycosylated(E3-Fc Non-gly) and glycosylated (E3-Fc Gly) iBio-CFB03, respectively(FIG. 35). The spectrum shows the monomeric subunit of the stablemultimer.

In-gel trypsin digestion of reduced and non-reduced bands of iBio-CFB03was performed to confirm the identity of the bands (FIG. 36). Forreduced conditions, band 1 corresponds to ˜35 kDa, band 2 corresponds to˜70 kDa, and band 3 corresponds to ˜100 kDa in accordance to themolecular weight marker. The tryptic peptides from each band were thenused to obtain the sequence coverage of iBio-CFB03. For non-reducedconditions, band 1 is the HMW multimer which never leaves the well.

In-gel tryptic digestion of iBio-CFB03 reduced band 1 resulted inseveral peptides from the E3 and Fc regions of the HMW multimer (FIG.37). In the iBio-CFB03 sequence given below, E3 peptide is highlightedin yellow. All the identified peptides are color coded in the sequencewith their respective masses highlighted in the MALDI spectrum (FIG.38).

Further Characterization of iBio-CFB03

Additional analyses are used to characterize iBio-CFB03. Negative-stainelectron microscopy is used to assess the uniformity of iBio-CFB03 anddetermine a molecular diameter from which an estimate of molecularweight can be derived. Characterization of the glycosylation pattern ofiBio-CFB03 also is performed.

Further mass spectrometric analyses will be performed usingquadrupole-time of flight (Q-TOF) interfaced to a HPLC to determine aprecise molecular weight of iBio-CFB03 prepared via separation on a RPHPLC column.

Potency Assay

The proposed assessment of potency for iBio-CFB03 takes a bimodalapproach to assess the activity of urokinase plasminogen activator (uPA)and the protein content of matrix metalloprotease-1 (MMP-1), twomediators involved in fibrosis. The uPA and MMP-1 assays are used toestimate the potency of iBio-CFB03 batches, based on their robustness inearlier mechanistic studies.

A number of experiments were performed to determine the proof of conceptand specific mechanism of action of iBio-CFB03. These studies andresults are described in detail herein. The majority of the proof ofconcept assays and the mechanism of action experiments were performedwith E4, an amidated form of the E3 peptide. The potency assays proposedbelow will be performed and qualified using recombinant iBio-CFB03.

uPA Activity Assay

Urokinase plasminogen activator, along with its receptor uPAR, initiatesa proteolytic cascade that results in the conversion of plasminogen toplasmin (Choong et al., Clin Orthop Relat Res. 2003; 415:S46-58). uPAhas an important function in extracellular proteolysis via its role inthe plasminogen system, which activates downstream enzymes and interactswith the extracellular matrix (ECM). Further, in select organs, uPA isimportant in stoichiometrically activating latent HGF, an anti-fibroticfactor (Naldini et al., J. Biol. Chemistry 1995; 270:603-611). uPA isproduced as an inactive protein that is proteolytically activated in theextracellular milieu or while bound to uPAR. Levels of uPA aresignificantly decreased in IPF patients (Gunther et al., Thrombosis andHaemostasis 2000; 83:853-860) and impaired plasminogen activation hasbeen reported in bronchoalveolar lavage fluid of patients with pulmonaryfibrosis. Multiple groups have demonstrated that uPA has anti-fibroticeffects in different organs and animal models. For example, inhaled uPAreduces airway remodeling in a murine model of asthma (Kuramoto et al.,Am. J Physiol.—Lung Cell. Mol. Physiol. 2009; 296:L337-346) andtransgenic expression of uPA in mouse lungs protects from fibrosis(Sisson et al., Human Gene Therapy 1999; 10:2315-2323; Sisson et al.,Am. J Physiol.—Lung Cell. Mol. Physiol. 2002; 283:L1023-1032; Idell, AmJ Respir Crit Care Med 2003; 168:1268-1269). Hsu et al have showndecreased levels of uPA in fibrotic lung tissues of patients with SScand IPF (Arthritis Rheum 2011; 63:783-794). Studies also havedemonstrated that E4 increases uPA levels and activity.

The assay is performed by incubating primary human fibroblasts withhuman recombinant TGF-β (10 ng/ml) or PBS as vehicle control withiBio-CFB03 (10 μg/ml) or DMSO (negative control). The uPA activity assayfrom Molecular Innovations (Human uPA Activity ELISA Kit Catalog#HUPAKT) is used to measure the activity of uPA in primary humanfibroblasts. The assay is an ELISA that detects uPA that is able to bindcovalently with human PA-1. The principle of the assay is thatfunctionally active uPA in samples forms a covalent complex with thebiotinylated human PA-1, which is bound to an avidin coated plate. BounduPA is then detected with an anti-uPA primary antibody (Ab), reactedwith horseradish peroxidase secondary Ab, followed by detection withtetramethylbenzidine (TMB) substrate at 450 nm. Inactive or complexeduPA does not bind and is not detected. The amount of color developmentis directly proportional to the concentration of active uPA in thesample. The concentration of active uPA in samples is determined from auPA standard curve.

MMP-1 Protein and Activity Assay

MMPs are zinc-dependent proteases that cleave ECM and other proteins(Amalinei et al., 2007) (Parks et al., Nature Reviews Immunology 2004;4:617-629). MMP-1 is the prototype interstitial collagenase 1 and is akey enzyme for degradation of fibrillar collagens in human tissues(Amalinei et al., Rom J Morphol Embryol 2007; 48:323-334). Evidence thatincreased activity of MMPs may resolve fibrosis comes from reports ofclinical trials using broad-acting metalloprotease inhibitors for thetreatment of various cancers where patients developed skin thickeningand joint contractures with MMP inhibitors (reviewed in Parks et al.,Nature Reviews Immunology 2004; 4:617-629), both of which occur in SSc.There is scientific evidence supportive of the therapeutic potential ofMMP-1, e.g. transient expression of MMP-1 is sufficient to attenuateliver fibrosis (Iimuro et al., Gastroenterology 2003; 124:445-458;transgenic mice expressing MMP-1 in mouse macrophages have decreasedcollagen deposition George, Clin Sci (Lond) 2012;122:83-92; Foronjy,Hypertension Res. 2008; 31:725-735); expression of human MMP-1 preventsmyocardial fibrosis (Foronjy et al., Hypertension Res. 2008;31:725-735); and recombinant MMP-1 resolves muscle fibrosis (Kaar etal., Acta Biomaterialia 2008; 4:1411-1420). Yamaguchi et al. havepreviously reported that E4 reduces levels of lysyl oxidase (LOX), anenzyme responsible for cross-linking and stabilizing collagen (SciTransl Med. 2012; 4:136ra171). These findings suggest that E4 weakensthe ECM by reducing LOX, thus facilitating its degradation by increasedMMP-1 and -3 levels. This also explains the ability of E4 to reduceongoing fibrosis via MMP-mediated matrix degradation. Activation ofMMP-1 and -3 via the plasminogen system is well documented (reviewed inGharaee-Kermani et al., Exp. Op. Invest. Drugs 2008; 17:905-916). MMP-1and -3 can also be induced by HGF (Jinnin et al., Nucleic Acids Research2005; 33:3540-3549; Kanemura et al., Hepatology Res. 2008; 38:930-939;Monvoisin et al., Int. J. Cancer 2002; 97:157-162; Dunsmore et al., J.Biol. Chem. 1996; 271:24576-24582; Huet et al., Biochem. Pharmacol.2004; 67:643-654).

Other studies revealed that E4 induces MMP-1 expression in human primaryfibroblast cells. Data also show that MMP-1 activity is increased insupernatants of fibroblasts treated with E4. MMP-1 levels are assayedusing a commercially available ELISA kit (Sigma-Aldrich Catalog#RAB0362-1KT) for the quantitative measurement of a MMP-1 protein levelsin primary fibroblasts as part of the potency testing for iBio-CFB03.Briefly, samples are pipetted into a 96-well plate coated withimmobilized MMP-1 antibody and after subsequent washing, form a covalentcomplex with the biotinylated human MMP-1 antibody. Bound MMP-1 isreacted with horseradish peroxidase followed by detection with TMBsubstrate solution. Inactive or complexed MMP-1 will not bind and willnot be detected. The amount of color development is directlyproportional to the concentration of MMP-1 in the sample, with theoverall intensity of the color being determined by absorbance at 450 nm.

MMP-1 activity levels in primary fibroblasts also is assayed using acommercially available antibody array kit (RayBiotech Catalog#AAH-MMP-1). Briefly, samples are pipetted and incubated in an 8-wellincubation tray containing antibody coated membranes. After subsequentwashing, the samples are incubated with biotinylated antibody. BoundMMP-1 is reacted with streptavidin conjugated with horseradishperoxidase followed by detection with detection buffers. The signalintensity is directly proportional to the concentration of active MMP-1in the sample as measured with a FluorChemR imaging system(ProteinSimple).

Stability Assessment

iBio-CFB03 is evaluated for real-time, real-condition stability underthe labeled conditions of storage, frozen conditions, and acceleratedconditions for at least three batches of plant-produced polypeptide (DS)and for each lot of the polypeptide as formulated for administration(DP). The duration of the stability studies for DS are 12 months forreal-time, real-condition and frozen conditions and 6 months foraccelerated conditions. The duration of the stability studies for DP are36 months for real-time, real-condition and frozen conditions and 6months for accelerated conditions. Testing frequency for all conditionsis done at release and every 3 months for DS. Testing frequency for DPin real-time, real-condition and frozen conditions is done at releaseand 3, 6, 9, 12, 18, 24 and 36 months, and at release and every 3 monthsfor accelerated conditions. Stability is assessed for the storageorientation of DP in upright, inverted and horizontal positions.

Nonclinical

Summary of Nonclinical Efficacy Studies

Studies performed with endostatin and chemically synthesized peptides E3and E4 provided further evidence of an anti-fibrotic effect. Thesechemically synthesized peptides do not form high molecular weightmultimers as seen with iBio-CFB03. Efficacy was observed in models ofSSc, with fibrosis induced in human skin ex vivo models by intradermalinjection of TGF-β, or in C57BL/6 mice by subcutaneous (s.c.)administration of either TGF-β or bleomycin. The E3 and E4 peptidesproduced through chemical synthesis attenuated fibrosis in these modelsupon intradermal or s.c. administration, demonstrated by decreases indermal thickness, collagen content, and hydroxyproline content. In thebleomycin-induced SSc mouse model, fibrosis reduction was achieved withboth prophylactic and interventional dosing regimens. These peptideswere also effective in IPF models induced by TGF-p treatment in primarycultures of human lung fibroblasts or instillation of bleomycin into thelungs of C57BL/6 mice. Peptides were administered to fibroblastcultures, or given orally, intravenously or intratracheally to mice.

Endostatin may be useful as an anti-fibrotic. Endostatin reversed injuryparameters in a rabbit ear scar model induced by mechanical wounding(Zhiyong et al., Int J Low Extrem Wounds 2012; 11(4):271-276; Ren etal., J. Zhejiang Univ. Sci. B. 2013; 14(3):224-230), in an IPF modelinduced by intra-tracheal (i.t.) bleomycin administration to rats (Wanet al., Respir Res. 2013; 14(1):56), in a liver fibrosis model inducedby administration of carbon tetrachloride to mice (Chen et al., Exp BiolMed. 2014; 239(8):998-1006), and in a diabetes/renal fibrosis modelinduced by streptozotocin administration to rats (Bai et al., Plos One.2014; 9(4):1-12).

In vivo studies are conducted with iBio-CFB03 at various doses anddosing schedules based on models previously established with thepeptides E3 and E4. Recombinant iBio-CFB03 also is used in the potencyassays.

TABLE 4 Summary of efficacy of endostatin and endostatin peptides infibrosis models Route of test Product product Model under testadministration Outcome Reference Normal Endostatin In vitroAdenovirally-expressed Yamaguchi et human endostatin markedly reducedal., Sci Transl lung fibronectin (FN) and type I Med. 2012; fibroblastscollagen αI chain (Col1α1) 4: 136ra171 protein levels in fibroblastspretreated with TGF-β. Human E4 peptide Ex vivo Single dose of E4,Yamaguchi et skin administered concurrently with al., Sci Translexplants, TGF-β, significantly decreased Med. 2012; TGF-β skin thicknessand attenuated 4: 136ra171 induced collagen increase. E4 also model ofameliorated fibrosis when SSc administered two days after TGF-βadministration. Mouse E3 & E4 In vivo Single dose of E3 or E4 Yamaguchiet TGF-β peptides peptide, administered al, Sci Transl dermalconcurrently with TGF-β, Med. 2012; model of significantly decreasedskin 4: 136ra171 SSc thickness. Mouse E4 peptide In vivo E4significantly decreased skin Yamaguchi et bleomycin s.c. thickness whenadministered as al, Sci Transl s.c. dermal a single doseprophylactically Med. 2012; model of and when given in a multiple 4:136ra171 SSc dose treatment regimen starting 3 days after initialexposure to bleomycin. Mouse E4 peptide In vivo E4 significantlydecreased Yamaguchi et bleomycin i.t. pulmonary fibrosis assessed al.,Sci Transl i.t. model microscopically and by Med. 2012; ofhydroxyproline/collagen 4: 136ra171 pulmonary quantification in lungsamples fibrosis when administered concomitantly or 3 days afterbleomycin. Mouse Biotinylated In vivo E4 significantly reduced Yamaguchiet bleomycin E4 peptide oral pulmonary fibrosis measured al, Sci Transli.t. model by hydroxyproline/collagen Med. 2012; of quantification inlung samples 4: 136ra171 pulmonary when administered fibrosisconcomitantly with bleomycin. Mouse iBio- In vivo iBio-CFB03significantly bleomycin CFB03 IV reduced pulmonary fibrosis i.t. modeloral compared to vehicle-treated of mice when administered by pulmonaryeither route concomitantly with fibrosis or 8 days following bleomycin.Mouse iBio- In vivo iBio-CFB03 significantly bleomycin CFB03 s.c.reduced dermal thickness as s.c. dermal assessed using the model ofhydroxyproline/collagen SSc quantification assay when administeredcontinuously for 7 days following bleomycin delivery. Human iBio- Exvivo Single dose of iBio-CFB03, skin CFB03 administered concurrentlywith explants, TGF-β, significantly attenuated TGF-β collagen increaseas assessed induced using the model of hydroxyproline/collagen SScquantification assay.

In Vivo Mouse Model of SSc

A mouse model of SSc has been established in which bleomycinadministered intratracheally can induce pulmonary fibrosis within a 12day timeframe (see FIG. 39). This model has been used to demonstrate theefficacy of iBio-CFB03, building on the experience using endostatinpeptides E3 and E4 to attenuate the development of fibrosis (Yamaguchiet al., Sci Transl Med. 2012; 4:136ra171). In this model, lung histologyand collagen content were measured with a Sircol assay 10 and 21 daysafter treatment with bleomycin (Yamaguchi et al., Sci Transl Med. 2012;4:136ra171).

Hydroxyproline is an amino acid unique to collagen, and is traditionallyemployed to quantify this protein. The method provides a quantitativedetermination of the total amount of hydroxyproline per mg wet tissue.The technique provides a high-throughput and accurate method to measurehydroxyproline, enabling the quantification of collagen in a variety offormats. In some instances, a hydroxyproline content assay should becomplemented by independent histological determinations of fibrosisusing a trichrome staining technique (or suitable alternative) to assessthe proportion and distribution of fibrotic tissue.

To evaluate tissue fibrosis/collagen deposition, a hydroxyprolinequantification assay was performed. Lung samples were removed anddigested overnight at 110° C. in 1 mL 6 N HCl. After neutralization with6 N NaOH, the pH was adjusted within the range of 6.0-9.0 and sampleswere mixed with 1 mL chloramine T solution (1.4% chloramine T, 10%isopropanol, 0.5 M sodium acetate, pH 6.0) for 20 minutes at roomtemperature. Following treatment with chloramine T solution, sampleswere mixed with 1 mL Erlich's solution (14.9% pdimethylaminobenzaldehyde, 70% isopropanol, 20% perchloric acid;Sigma-Aldrich) and incubated for 15 minutes at 65° C. Aliquots weretransferred to 96-well plates, and absorbance was measured at 570 nm.Collagen content was calculated by comparison with a standard curvegenerated with cis-4 hydroxy-L-proline (Sigma-Aldrich), using theconversion factor of 1 μg hydroxyproline corresponding to 6.94 μg. Thisassay has been used in the studies described below using iBio-CFB03 andis used in non-clinical studies in this mouse model.

Intravenous and Oral Administration of iBio-CFB03 in Bleomycin MouseModel

C57BL/6 mice were given bleomycin i.t. in conjunction with iBio-CFB03intravenous (IV) administration or followed by IV administration ofiBio-CFB03 eight days after bleomycin. The mice were sacrificed at day12 and lung samples were taken for both H&E staining (FIG. 40) andhydroxyproline assay (FIG. 41). A diagram of the experimental design canbe found in FIG. 39. Compared to mice treated with bleomycin only, micetreated repeatedly with iBio-CFB03 IV at 100 μg or 200 μg/mouse hadreduced pulmonary fibrosis as assessed using hydroxyproline assays.Intravenously administered iBio-CFB03 at either dose appeared to havesimilar efficacy to orally administered iBio-CFB03 at 50 μg. iBio-CFB03administered eight days after bleomycin treatment (both orally andintravenously) also appeared to reduce fibrosis. However only 3 micewere treated in each of these groups. These experiments were thereforerepeated with iBio-CFB03 administered IV at 200 μg/mouse (n=9) or orallyat 50 μg/mouse (n=9) (FIG. 42). Mice treated IV with 100 μg/mouseiBio-CFB03 had reduced pulmonary fibrosis compared to mice treated withbleomycin only. Administration either by IV or oral route appeared tohave similar efficacy.

iBio-CFB03 ameliorated bleomycin-induced pulmonary fibrosis in vivo,whether administered concomitantly or after a fibrotic trigger(represented as d8). 1×PBS was used as vehicle. Mean collagen values arepresented as micrograms per lung. iBio-CFB03 was administered eitherorally (oral) or intravenously (IV). Error bars in FIG. 41 representstandard deviation of the mean. P-values were calculated usingKruskal-Wallis test, followed by Mann-Whitney U test.

Osmotic Pump Administration of iBio-CFB03 in Mouse Dermal Model of SSc

C57BL/6 mice were given 33 mU of bleomycin for seven days usingmini-osmotic pumps surgically implanted on the backs of the mice todeliver bleomycin subcutaneously. On day 7, the pumps were removed andreplaced with pumps containing 460 μg of iBio-CFB03 administered s.c.for an additional seven days. Mice were sacrificed at day 35 and skinfrom the back on the opposite side of the pump site was excised,formalin-fixed and embedded in paraffin. Skin sections were stained withH&E (FIG. 43) and dermal thickness (FIG. 44) was measured. Mice treatedwith iBio-CFB03 had reduced dermal thickness as assessed usinghydroxyproline assays.

Ex Vivo Human Skin Model of SSc

Efficacy of iBio-CFB03 was assessed in human skin models of SSc inducedby injection of TGF-β. TGF-β induces pathological changes in skincharacteristic of SSc. iBio-CFB03 attenuated TGF-β induced fibrosis inhuman skin when administered concurrently with TGF-β. When administeredconcurrently with 10 ng TGF-β, a single intradermal dose of 100 μgiBio-CFB03 prevented the increased collagen when evaluated one week postdosing via hydroxyproline assay (FIG. 45).

Human skin was obtained from abdominal surgery, trimmed, fat cleaned,and cut into ˜1×1 inch pieces. Pieces were maintained in organ culturein an air-liquid interface. Pieces were injected with vehicle (V), TGF-β(T) or TGF-β+iBio-CFB03 (T+iBio-CFB03) in a volume of 100 μl. Doses:Vehicle=100 μl of 1×PBS; T=10 ng of TGF-β; T+iBio-CFB03=10 ng TGF-β+100μg iBio-CFB03. After one week (7 days), 3 mm punches of skin wereobtained and hydroxyproline measured in the skin punches. The datarepresent n=3/group. * denotes p<0.05.

Justification of E4 Data Supportive of iBIO-CFB03

Evaluation of the efficacy and mechanism of action of C-terminalendostatin peptides in preclinical fibrosis models has been performedusing chemically-synthesized E3, and the closely related peptide, E4. E4differs from E3 by an addition of a C-terminal amide to stabilize the E3peptide. Efficacy and mechanistic studies using recombinant iBio-CFB03are done to confirm the anti-fibrotic activity previously demonstratedwith chemically-synthesized E3 and E4. iBio-CFB03 improves thesolubility, stability and purification of E3/E4, and has shown promisingresults in further pre-clinical testing.

Mechanism of Action Studies with E4 Peptide

Several experiments have been performed to identify the mechanisms bywhich E4 exerts its anti-fibrotic activity. Real-time quantitative PCR(qPCR)-based arrays were performed on RNA extracted from primary humanlung fibroblasts or mouse lung tissue stimulated with various fibrotictriggers in the absence and presence of E4 treatment in order toidentify genes influenced by the endostatin peptide.

The arrays identified several genes that have been previously implicatedin fibrosis to be influenced by E4 treatment. These genes includedconnective tissue growth factor (CTGF), insulin-like growth factorbinding protein (IGFBP)-3, matrix metalloproteases (MMP)-1 and MMP-3,urokinase plasminogen activator (uPA), plasminogen activator inhibitor(PAI)-1, and hepatocyte growth factor (HGF) with levels either decreasedor increased in the presence of E4 as shown in TABLE 5.

TABLE 5 Genes regulated by E4 Gene Model Method Effect of E4 peptideCTGF Primary human RT-qPCR Decrease fibroblasts treated with bleomycin(HuF-bleo) or TGF-β (HuF-T) as fibrogenic trigger IGFBP-3 HuF-T RT-qPCRDecrease Protein expression levels uPA HuF-T RT-qPCR Increase Proteinexpression levels Enzyme activity PAI-I HuF-T RT-qPCR Decrease MouseProtein expression bleomycin model of pulmonary fibrosis HGF HuF-TRT-qPCR Increase Mouse Protein expression bleomycin model of pulmonaryfibrosis MMP-1 HuF-T RT-qPCR Increase Protein expression levelsZymography MMP-1 activity assay MMP-3 HuF-T RT-qPCR Increase Proteinexpression levels Zymography

Summary and Model for Mechanism of Action

In summary, E4, a peptide derived from endostatin, exerts anti-fibroticeffects in vitro, in vivo and ex vivo after application of two differentfibrotic triggers, bleomycin and TGF-β (Yamaguchi et al., Sci TranslMed. 2012; 4:136ra171). E4 reduces levels of the pro-fibrotic factorsCTGF and IGFBP-3 and induces increased levels and activity of MMP-1 andMMP-3. Further, E4 activates the plasminogen system, increases uPAlevels and activity, reduces levels of the uPA inhibitor PAI-1, andinduces conversion of pro-HGF to its active chains. E4 exertsanti-fibrotic effects via down-modulation of PM-1 and an increase of uPAactivity, leading to HGF activation, decreased fibrotic intermediariesCTGF and IGFBP-3, and increased ECM degradation through induction ofactive MMP-1 and MMP-3 production. Target specificity is summarized inthe schematic shown in FIG. 46.

PK and Toxicology Studies Pharmacokinetic Studies

Method(s) of detection for iBio-CFB03 in biological specimens, includingserum and skin, are established. Pharmacokinetics and metabolism ofiBio-CFB03 are evaluated in animals. Initial, exploratory studies areconducted in mice to determine serum concentration of iBio-CFB03, andbegin to establish a relationship between an efficacious IV dose andcirculating drug levels. Exploratory PK studies in rats and rabbits arealso done to examine potential species differences in iBio-CFB03absorption, and to determine serum concentration of iBio-CFB03 in thetwo species proposed for acute and chronic toxicology studies. Morecomprehensive PK/ADME assessments of iBio-CFB03 in rats and rabbits areconducted to characterize PK profiles and dose linearity. Some of thePK/ADME assessments are conducted in the toxicokinetic portion of theproposed toxicology studies.

The commercially available polyclonal enzyme immunoassay (EIA) 96-wellplate assay (Accucyte, Cytimmune Sciences Inc., College Park, Md.) usedfor assessing endostatin PK (Thomas et al., J Clin Oncol. 2003;21(2):223-231) is tested for its ability to detect iBio-CFB03. Inaddition to the published method in Thomas et al., the feasibility ofusing an immunoglobulin Y (IgY) antibody for immunological studies isinvestigated. IgY antibodies produce less cross-reactivity andbackground against a mammalian antigen than systems in which theantibody/antigen complex are both from mammalian sources. Additionally,murine monoclonal antibodies are developed against iBio-CFB03 for thepurpose of constructing a sandwich ELISA for PK analysis and otherquantification needs in tissue or biofluids. As an additional technologyto generate iBio-CFB03-specific immune reagents, chicken spleen cellsare used for generating polyclonal IgY anti-iBio-CFB03 antibodies asstarting material to generate single-chain variable binding fragments(scFv) or recombinant monoclonal IgY antibodies that could be engineeredto generate binding and detection reagents.

If a sensitive and specific sandwich ELISA for detection of iBio-CFB03in tissue and biofluids is not possible, a MS/MS-based assay isdeveloped for selective detection of endostatin-related peptide usingmultiple reaction monitoring, which provides a high level of sensitivityfor specific peptide fragments. Ideally, such an assay focuses onpeptide fragments unique to the iBio-CFB03 to distinguish it from thenatural level of endostatin or its precursor collagen molecule. Thesuccess of this approach also depends on being able to detect suchfragments in complex biological samples, and involves specifictissue/biological fluid preparation and separation protocols to“decomplex” the sample to maximize detection and reproducibility.

In some cases, the methods for characterization of iBio-CFB03 areadopted for bioanalytical assessments. These methods includematrix-assisted laser desorption/ionization (MALDI), immunoassays, andLC/MS-MS.

Following establishment of an adequate bioanalytical method foriBio-CFB03, initial pilot studies are conducted in mice to assess IVadministration of iBio-CFB03, and to begin to establish a possiblerelationship between efficacious dose levels identified from the proofof concept mouse studies and levels of drug in serum and target tissue.Pilot PK studies in rats and rabbits also are used to establish thatiBio-CFB03 is efficacious in the two species used for acute and chronictoxicology studies. Additionally, potential species differences iniBio-CFB03 absorption and PK profiles are identified.

After exploratory studies, more comprehensive PK/ADME assessments ofiBio-CFB03 in rats and rabbits are carried out characterize PK profilesand dose linearity, and to support dose selection in the Phase 1 study.Designs for these studies include blood collection pre-dose, immediatelypost-dose, and up to 120 h post-dose. Groups of animals are staggered orslated for terminal collection (rats only) during sampling depending onspecies and frequency of sampling. Skin also is collected from ratsslated for terminal collection. Serum and skin levels of iBio-CFB03 areassessed.

Additional PK/ADME assessments are conducted in the toxicokineticportion of the toxicology studies, and the PK of iBio-CFB03 is evaluatedfollowing single and multiple (daily/weekly) dose administration.

Single Dose Toxicology

Non-GLP single dose toxicology studies in Sprague-Dawley rats and NewZealand White rabbits are done to facilitate dose selection for GLPanimal toxicology studies, and to support initiation of testing inhealthy volunteers in the Phase 1 study.

Non-GLP Acute Rat Toxicology Study

Toxicity of iBio-CFB03 is assessed in male and female adultSprague-Dawley rats following a single IV administration. Three doselevels of IV administered iBio-CFB03 are evaluated, and compared to onegroup of rats given a single IV administration of vehicle as control.Three rats per group and per gender are used based on standard practicefor this type of study in order to provide adequate determination ofpotential toxicity and statistical differences among study groups.

Evaluations include assessments of mortality, morbidity, clinicalobservations, and body weight changes. Blood samples are collected forhematology, clinical chemistries, and coagulation parametersassessments. Organs are weighed and examined for any gross pathologicalchanges. Histopathology is performed on organs pending gross necropsyfindings.

Determination of iBio-CFB03 in skin specimens and serum are used toevaluate systemic and target organ exposure. MMP-1 and uPA levels inskin specimens and serum are determined for baseline assessments ofthese SSc biomarkers.

Rats are used for these tests since they are one of the model speciesfor the assessment of toxicity, and one of the species recommended forthis purpose by toxicity testing guidelines of all regulatory agencies.

Non-GLP Acute Rabbit Toxicology Study

Toxicity of iBio-CFB03 also is assessed in New Zealand White male andfemale adult rabbits following a single IV administration. Three doselevels of intravenously administered iBio-CFB03 are evaluated, andcompared to one group of rabbits given a single IV administration ofvehicle as control. Two rabbits per group and per gender are used, basedon standard practice for this type of study in order to provide adequatedetermination of potential toxicity and statistical differences amongstudy groups.

Evaluations include assessments of mortality, morbidity, clinicalobservations, and body weight changes. Blood samples are collected forhematology, clinical chemistries, and coagulation parametersassessments. Organs are weighed and examined for any gross pathologicalchanges. Histopathology is performed on organs pending gross necropsyfindings.

Determination of iBio-CFB03 in skin specimens and serum are used toevaluate systemic and target organ exposure. MMP-1 and uPA levels inskin specimens and serum are determined for baseline assessments ofthese SSc biomarkers.

Rabbits are used for these tests as they are another of the modelspecies for the assessment of toxicity and one of the speciesrecommended for this purpose by toxicity testing guidelines of allregulatory agencies.

Repeat Dose Toxicology

Twenty-eight (28) day repeat dose GLP toxicology studies are conductedin Sprague-Dawley rats and New Zealand White rabbits to supportinitiation of testing in healthy volunteers in the Phase 1 study.

Twenty-Eight Day Repeat Dose GLP Rat Toxicology Study

Toxicity of iBio-CFB03 is assessed in male and female adultSprague-Dawley rats following 28 consecutive days of every other day(3×/week) IV administration. Three dose levels of IV administerediBio-CFB03 are evaluated, and compared to one group of rats given asingle IV administration of vehicle as control. Dose selection are basedon findings from the PK rat studies, as well as maximumtolerated/feasible and NOAEL doses identified in pilot and single doserat toxicology studies.

In addition to a main study portion, satellite groups of animals areincluded for 28 day recovery period and toxicokinetic analyses.

The study design is summarized in TABLE 6.

TABLE 6 Design for the 28 Day Repeat Dose GLP Rat Toxicology Study DoseNumber of Animals Level Main Study Recovery Satellite TK Group Treatment(mg/kg) Males Females Males Females Males Females 1 Control 0 10 10 5 52 iBio- 2.5 mg/kg 10 10 6 6 CFB03 3 iBio-   5 mg/kg 10 10 6 6 CFB03 4iBio-  10 mg/kg 10 10 5 5 6 6 CFB03

The number of rats per group and per gender are based on standardpractice for this type of study in order to provide adequatedetermination of potential toxicity and statistical differences amongstudy groups.

Evaluations include assessments of mortality, morbidity, clinicalobservations, and body weight changes. Blood samples are collected forhematology, clinical chemistries, and coagulation parametersassessments. Organs are weighed and examined for any gross pathologicalchanges. Histopathology is performed on organs pending gross necropsyfindings.

Determination of iBio-CFB03 levels in skin specimens and serum are usedto evaluate systemic and target organ exposure. MMP-1 and uPA levels inskin specimens and serum are determined for baseline assessments ofthese SSc biomarkers.

Rats are used for these tests as they are one of the model species forthe assessment of toxicity and one of the species recommended for thispurpose by toxicity testing guidelines of all regulatory agencies.

Twenty-Eight Day Repeat Dose GLP Rabbit Toxicology Study

Toxicity of iBio-CFB03 is assessed in New Zealand White male and femaleadult rabbits following 28 consecutive days of once daily IVadministration. Three dose levels of IV administered iBio-CFB03 areevaluated and compared to one group of rabbits given a single IVadministration of vehicle as control. Dose selection is based onfindings from the PK rabbit studies, as well as maximaltolerated/feasible and NOAEL doses identified in pilot and single doserabbit toxicology studies.

In addition to a main study portion, satellite groups of animals areincluded for 28 day recovery period and toxicokinetic analyses.

The study design is summarized in TABLE 7.

TABLE 7 Design for the 28 Day Repeat Dose GLP Rabbit Toxicology StudyNumber of Animals Dose Recovery Satellite TK Level Main Study (optional)(optional) Group Treatment (mg/kg) Males Females Males Females MalesFemales 1 Control 0 4 4 2 2 2 iBio- 2.5 mg/kg 4 4 2 2 CFB03 3 iBio-   5mg/kg 4 4 2 2 CFB03 4 iBio-  10 mg/kg 4 4 2 2 2 2 CFB03

The number of rabbits per group and per gender are based on standardpractice for this type of study in order to provide adequatedetermination of potential toxicity and statistical differences amongstudy groups.

Evaluations include assessments of mortality, morbidity, clinicalobservations, and body weight changes. Blood samples are collected forhematology, clinical chemistries, and coagulation parametersassessments. Organs are weighed and examined for any gross pathologicalchanges. Histopathology is performed on organs pending gross necropsyfindings.

Determination of iBio-CFB03 in skin specimens and serum are used toevaluate systemic and target organ exposure. MMP-1 and uPA levels inskin specimens and serum are determined for baseline assessments ofthese SSc biomarkers.

Rabbits are used for these tests as they are a model species for theassessment of toxicity and one of the species recommended for thispurpose by toxicity testing guidelines of all regulatory agencies.

Nonclinical Development Plan

Nonclinical development of iBio-CFB03 includes evaluating efficacy infibrosis models to confirm the previously demonstrated anti-fibroticactivity demonstrated with structurally related endostatin peptides.Studies exploring the mechanism of action of iBio-CFB03 also are used tofurther characterize its anti-fibrotic activity. In addition,pharmacokinetics of iBio-CFB03 are evaluated.

Immunogenicity of iBio-CFB03 is evaluated in rats and rabbits foranti-drug antibodies. Initial screening assays are conducted and samplestesting positive in the screening assay are re-tested using aconfirmatory assay. A neutralization assay also is performed to measureany neutralizing antibody activity.

Potential toxicity of iBio-CFB03 is evaluated in rats and rabbits.Initial pilot studies are used to explore the ability to achieve amaximal tolerated dose, or otherwise, establish a maximal feasible dose,based on animal tolerability and compound availability following anacute, single dose administration of iBio-CFB03. Repeat dose studies inrats and rabbits are also conducted to explore potential toxicity ofiBio-CFB03 following 28 days of repeat dose administration (every otherday; 3×/week) under GLP compliance. Pending outcome of single dose and28 day GLP repeat dose studies, chronic toxicology studies are conductedin both rodent and non-rodent species in 6 month repeat dose GLP studiesto support chronic use.

Clinical

In a Phase 1 PK and PD study of recombinant human endostatin in patientswith advanced solid tumors, it was demonstrated that endostatin isessentially free of significant drug-related toxicity and was welltolerated when given daily as a 1-hour intravenous infusion at doses upto 300 mg/m2 (Thomas et al., J Clin Oncol. 2003; 21(2):223-231).

An open label, two segment Phase 1 trial is conducted in normal healthyvolunteers (NHV) and SSc patients to assess the safety, immunogenicity,and pharmacokinetics of intravenously administered iBio-CFB03. Segment Ais a single-dose, dose escalating design. After the completion of thissegment, a Data Safety Monitoring Board (DSMB) reviews the PK and safetydata to determine the Maximum Tolerated Dose (MTD). SSc patients areenrolled in Segment B of the study for repeat dose exposure at the MTDfrom the first segment. The waiting period between first and seconddoses (1 week) and/or dosing schedule (other day after dose 2) areadjusted as needed in Segment B.

The maximum dose for evaluation in Segment A of the study, 5 mg, wasselected as a possible therapeutic dose based on results from the proofof concept studies in the mouse-bleomycin model. In those studies, theminimum dose at which significant effects of iBio-CFB-3 were observedwas 20 μg per animal. Scaling this to a human equivalent dose (HED)using the conversion factor provided in FDA Guidance to Industry:Estimating the Maximum Safe Starting Dose in Initial Clinical Trials forTherapeutics in Adult Healthy Volunteers (July 2005), gives:

[20 μg/20 g mouse]*0.081*60 kg human=4.8 mg per dose

The toxicology studies are designed to provide a safety factor of 10fold excess above this maximum human dose for the Phase 1 clinicaltrial.

Immunogenicity Sampling

Because iBio-CFB03 is a therapeutic protein, volunteers enrolled in theclinical study are monitored for the incidence of anti-drug antibodies(ADA). Serum samples for ADA screening are collected prior toadministration of the first dose, prior to the administration of thefinal dose (Segment B participants only), 14 days, and 28 days after theadministration of the final dose.

Pharmacokinetics Sampling

In Segment A of the study, serum samples for assessment of iBio-CFB03pharmacokinetics (Cmax, AUC0 ∞, serum concentration, eliminationconstant, clearance) are collected at the following time points:

-   -   0 min (pre-dose baseline)    -   5 min    -   15 min    -   30 min    -   1 h    -   2 h    -   6 h    -   12 h

In Segment B of the study, Dose 1 PK serum samples are collected fromparticipating SSc patients according to the same sampling plan describedabove. For subsequent study doses, a single serum PK sample is collectedat the timepoint closest to Tmax¬ observed in Segment A.

Pharmacologic Activity

SSc patients enrolled in Segment B are assessed for possiblepharmacological activity of iBio-CFB03 by serial skin biopsy. Skinbiopsies are performed at baseline, four weeks and twelve weeks. Twobiopsies are taken at baseline, one from each arm. The four week biopsyis taken on one arm and the twelve week biopsy on the other. Two skinbiopsies are taken on each individual arm in a FIG. 8 pattern. One isused for histology with appropriate staining to assess any changes inskin thickness or collagen organization (e.g., H&E, Mason's trichrome,Verhoeff-Van Gieson) and the other for a hydroxyproline assay.

Clinical Development Plan

An open label Phase 2a study is conducted to assess the safety, PK andpreliminary efficacy of the iBio-CFB03 in diffuse SSc patients viaintravenous administration. This study is a repeat-dose open-label trialdesigned to evaluate the dose effect and dose tolerance relationships aswell as to determine the appropriate dosing regimen. The dose levelschosen for this Phase 2a study are chosen based on data from the Phase 1study and non-clinical toxicology studies performed with the same routeof administration. Subsequent to the Phase 2a study, a conduct 3-6 monthPhase 2b study is conducted using an intravenous administration ofiBio-CFB03 to assess safety and efficacy, followed by aplacebo-controlled Phase 3 study of iBio-CFB03 in diffuse SSc patients.A summary of the clinical development plan (CDP) is shown in TABLE 8.

TABLE 8 Clinical Development Plan Secondary and Sample PrimaryExploratory Phase Description Population size Objectives ObjectivesPhase PK, Segment A: Segment Safety and Evaluate the 1 immunogenicityNormal healthy A: 20 tolerability of pharmacokinetic and safety studyadult subjects intravenously profile of iBio- (1 month) (>18 years)Segment administered CFB03 Segment A: volunteers B: 20 iBio-CFB03Determine dose Single dose, Segment B: subjects Evaluate needed to reachdose escalating Diffuse SSc immunogenicity potentially design patientsof iBio-CFB03 therapeutic serum Segment B: levels Repeat dose,Assessment of fixed dose level potential on pharmacodynamic biomarkersin serum/plasma and skin Phase Multiple doses, Patients TBD Safety andDefine dose effect 2a dose escalating, (diffuse SSc; tolerability of anddose tolerance PK/PD and early stage <18 intravenously relationshipssafety study months) administered Determine (~3 months) iBio-CFB03appropriate dose Pharmacokinetic and dosing profile regimen PreliminaryPreliminary efficacy based on efficacy data based Rodnan skin on Rodnanskin scores scores Phase High vs. low Patients TBD Efficacy (high vs.Skin biopsies to 2b dose, efficacy (diffuse SSc; low dose) determinestudy early lung or potential markers (3-6 months) early kidney (uPA;MMP-1) involvement) Exploratory serum biomarkers Risk/benefit analysisExploratory: lung improvement Phase Randomized Patients TBD Efficacyagainst Efficacy 3 controlled (diffuse SSc; comparator Skin biopsies;look trial(s) early stage, 18 (placebo) at various markers months)Exploratory: lung improvement

Development of an Oral Route of Administration

Preliminary data suggests that oral dosing with iBio-CFB03 is at leastas efficacious as an IV route of administration. Based on these data andthe benefits of an oral route of administration for what will likely bechronic use, an oral formulation of iBio-CFB03 is developed. If it isdetermined that an oral formulation is beneficial to the program, thestudies outlined in TABLE 9 are performed prior to introducing the oralformulation into clinical studies.

TABLE 9 Non-Clinical Studies to Support Oral Administration Study ModelDosing Route Objective Oral Mouse and Oral and IV Determine serumbioavailability rats levels of orally administered iBio- CFB03 comparedto IV administration Food effect Rats Oral Determine the effect of foodon the pharmacokinetics of iBio-CFB03 90 Day Repeat Rats Oral Assessmentof Dose Toxicology potential toxicity of iBio-CFB03; recovery, and TKparameters

Example 12 Oral Delivery Fc Fusion Proteins

E3-Fc (END-25), E3-linker-Fc (END-26), and C67A E3-Fc (END-55) lysateswere run on an SDS-PAGE gel, imaged for total protein (FIG. 47, leftpanel), and probed with an anti-Fc antibody by Western blot (FIG. 47,right panel).

Equal amounts of purified END-55 from Novici (PEG purified) or Caliber(Protein A purified) were digested in Simulated Gastric Fluid (SGF) for0, 5, 30, 45, 60, or 300 seconds. The reaction was stopped with 160 mMsodium carbonate. Samples were then run on a 4-20% SDS-PAGE gel andimaged on a stain-free gel imager for total protein (FIG. 48, leftpanel). Gel was then transferred to nitrocellulose and probed witheither a chicken anti-E3 antibody (FIG. 48, middle panel) or anti-humanFc antibody (FIG. 48, right panel) for Western blot. E3 plus probablythe hinge region of Fc does appear to be liberated from the Fc molecule,but is largely degraded by the 300 second time point. Differencesbetween the Caliber and Novici samples appear to be a ˜14 kD band whichis more prominent in the early time points for Caliber material and lessglycosylated E3-Fc in the Caliber samples. The ˜10 kD E3-positive bandis slightly weaker in the Caliber samples. All images are scaled andaligned with each other.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An isolated polypeptide, wherein the polypeptide: a) comprises SEQ IDNO:14; and b) has anti-fibrotic activity when administered to a subjectin need thereof.
 2. The isolated polypeptide of claim 1, furthercomprising a secretory sequence.
 3. The isolated polypeptide of claim 1,further comprising a peptide tag.
 4. The isolated polypeptide of claim3, wherein the peptide tag is a 6His tag.
 5. The isolated polypeptide ofclaim 3, wherein the peptide tag comprises a KDEL polypeptide.
 6. Theisolated polypeptide of claim 1, wherein the polypeptide furthercomprises an Ala-Ser-Lys sequence at the C-terminal end of SEQ ID NO:14.7. The isolated polypeptide of claim 1, further comprising an IgG Fcdomain.
 8. The isolated polypeptide of claim 7, wherein the IgG Fcdomain is an IgG1 Fc domain.
 9. The isolated polypeptide of claim 8,wherein the IgG1 Fc domain is a human IgG1 Fc domain.
 10. The isolatedpolypeptide of claim 1, further comprising amino acids 27 to 43 of SEQID NO:17.
 11. The isolated polypeptide of claim 1, wherein thepolypeptide comprises SEQ ID NO:16.
 12. The isolated polypeptide ofclaim 1, wherein the polypeptide comprises SEQ ID NO:17. 13-14.(canceled)
 15. An isolated polynucleotide encoding the polypeptide ofclaim 1, wherein the polynucleotide is operably linked to a heterologouspromoter.
 16. An expression vector comprising the isolatedpolynucleotide of claim
 15. 17-18. (canceled)
 19. An Agrobacteriumtumefaciens cell comprising the expression vector of claim
 16. 20.(canceled)
 21. A method for making the polypeptide of claim 1 havinganti-fibrotic activity, the method comprising: (a) introducing into aplant a plant viral vector that includes a polynucleotide encoding thepolypeptide of claim 1 having antifibrotic activity; and (b) maintainingthe plant under conditions and for a time sufficient that thepolynucleotide is expressed in at least some plant cells.
 22. The methodof claim 21, wherein the introducing comprises vacuum infiltration. 23.The method of claim 21, further comprising harvesting the plant, whereinthe plant comprises the polypeptide having anti-fibrotic activity. 24.The method of claim 21, further comprising extracting the polypeptidehaving anti-fibrotic activity from the plant.
 25. The method of claim24, further comprising purifying the polypeptide having anti-fibroticactivity. 26-35. (canceled)